Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-trisphosphate

The present study was carried out to evaluate the effects of different acti vation protocols, enucleation methods, and culture media on the development of parthenogenetic and nuclear transfer (NT) rabbit embryos. Electroporati on of 25 mM inositol 1,4,5-trisphosphate (IP3) in calcium- and magnesium-fr ee PBS immediately induced a single intracellular calcium transient in 6 ou t of 14 metaphase II-stage rabbit oocytes evaluated during a 10-min recordi ng period. The percentage of oocytes treated with IP3 followed by 6-dimethy laminopurine (IP3 + DMAP) that cleaved (83.9%) and reached the blastocyst s tage (50%) was significantly higher (p < 0.05) than those activated with mu ltiple pulses (61.6% and 30.1%, respectively) or treated with ionomycin + D MAP (52.9% and 5.7%, respectively). Development of IP3 + DMAP-activated rab bit oocytes and in vivo-fertilized zygotes in different culture media was s tudied. Development of activated oocytes to the blastocyst stage in Earle's balanced salt solution (EBSS) supplemented with MEM nonessential amino aci ds, basal medium Eagle amino acids, 1 mM L-glutamine, 0.4 mM sodium pyruvat e, and 10% fetal bovine serum (FBS) (EBSS-complete) (40.6%) was significant ly higher (p ( 0.05) than those that developed in either Dulbecco's Modifie d Eagle's medium (DMEM)/RPMI + 10% FBS (15.5%) or CR1aa + 10% FBS (4%) medi um. In addition, 100% of in vivo-fertilized rabbit zygotes developed to the blastocyst st;age in EB SS-complete. A third set of experiments was carrie d out to study the efficiency of blind versus stained (Hoechst 33342) enucl eation of oocytes. Twenty-nine of 48 blind enucleated and IF, + DMAP-activa ted oocytes cleaved (60.4%), and 15 (31.2%) subsequently reached the blasto cyst stage, whereas 9 of 52 oocytes enucleated using epifluorescence (17.3% ) cleaved, and none of these reached the blastocyst stage. When the above p arameters that yielded the highest blastocysts were combined in an NT exper iment using adult rabbit fibroblast nuclei, 72.2% (39 of 54) of the fused n uclear transplant embryos cleaved and 29.6% (16 of 54) reached the blastocy st stage.