Electrofusion of two-cell bovine embryos for the production of tetraploid blastocysts in vitro

• Published in: Molecular reproduction and development 2000-07, Vol.56 (3), p.372-377
• Main Authors: Curnow, E.C., Gunn, I.M., Trounson, A.O.
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Blastocyst - physiology; Cattle; Cell Fusion; chimera; Early stages. Segmentation. Gastrulation. Neurulation; Embryology: invertebrates and vertebrates. Teratology; Fundamental and applied biological sciences. Psychology; karyotype; Karyotyping; mosaic; tetraploidy; Time Factors; Zygote - physiology
• ISSN: 1040-452X; 1098-2795
• DOI: 10.1002/1098-2795(200007)56:3<372::AID-MRD7>3.0.CO;2-W

Tetraploid bovine blastocysts were produced experimentally by electrofusion of in vitro matured and fertilized, zona‐enclosed two‐cell embryos (33–35 hr after initiation of sperm–egg incubation) using three fusion protocols. Field strengths of 1.0, 1.4, and 2.4 kV/cm were tested and the rate of fusion, subsequent cleavage, and blastocyst development were measured for each. High rates of fusion (76.5% ± 2.8%), cleavage (72.5% ± 7.4%) and blastocyst development (56.1% ± 6.4%) were achieved with the application of 1.4 kV/cm as a single 100‐μs pulse. Embryos were scored 30 and 60 min after stimulation for fusion. No time effect for fusion, cleavage, or blastocyst development was observed. Chromosome preparations of day 7 blastocysts revealed 12.5% of fused embryos were tetraploid. This is a significant increase from that found in nonfused embryos where spontaneous tetraploidy did not occur. An electrical stimulus of 1.0 kV/cm applied as two 50‐μs pulses produced significantly less one‐cell embryos (64.2% ± 3.0%) compared to 1.4 kV/cm while cleavage (79.9% ± 3.4) and blastocyst development (44.6% ± 4.0%) were not different from that for unexposed control embryos (89.5% ± 2.3% and 57.2% ± 3.2%, respectively). Embryos fused at 2.4 kV/cm applied as a single 30‐μs pulse (69.7% ± 5.7%) showed significantly lower cleavage (72.1% ± 3.7%) and blastocyst rates (40.2% ± 4.6%) compared to the unexposed control. Mol. Reprod. Dev. 56:372–377, 2000. © 2000 Wiley‐Liss, Inc.