Phytohemagglutinin improves efficiency of electrofusing mammary gland epithelial cells into oocytes in goats

Abstract 

The objective was to investigate the effect of phytohemagglutinin (PHA) on the fusion of mammary gland epithelial (MGE) cells into enucleated oocytes in goats. The toxicity of PHA was evaluated by testing its effect on the development of parthenogenetic caprine oocytes. The effective dose and duration of PHA treatment (100μg/mL, 20min incubation) was selected and used to compare fusion efficiency and embryo development following nuclear transfer. Two electrofusion protocols, chamber fusion (CF) and pressurized microelectrode fusion (pMEF), were also compared, when couplets were treated with and without PHA (100μg/mL, 20min). Fusion rate of couplets increased from 52.8 to 74.0% for the CF protocol (P<0.05), but was not significantly different for the pMEF protocol (72.7% vs. 78.1%) after PHA treatment. There were no significant differences between treated group and control in rates of subsequent cleavage or blastocyst development. Following transfer of the cloned blastocysts derived from the PHA-treated group and the control group into synchronized recipients, pregnancy rates (Day 30) were not significantly different between treated group and control (28.6% vs. 25.0%). However, all recipients aborted within 120d, microsatellite DNA analyses confirmed that the aborted fetuses were genetically identical to the donor goat. In conclusion, the fusion rate of caprine MGE cell couplets was improved by pre-incubating couplets in medium containing 100μg/mL PHA prior to electrical pulsing, and embryos derived from PHA treatment established early pregnancies.

Keywords: Electrofusion, Mammary gland epithelial cells, Nuclear transfer, Phytohemagglutinin, Goat