DEVELOPMENT OF PARTHENOGENETIC AND CLONED OVINE EMBRYOS - EFFECT OF ACTIVATION PROTOCOLS

Preliminary experiments carried out on ovine oocytes were designed to establish correlations between activation protocols and subsequent rat es of embryonic development. The best activation protocols were therea fter used in studies on ovine parthenogenesis and cloning. The first s tudy established that chemical activators induce pronuclear developmen t at a slightly higher rate than physical activation (ionomycin, 96%; ethanol, 95%; electro activation, 80%). Inhibition of second polar bod y extrusion and one single pronucleus were observed in the majority of the oocytes (similar to 90%) treated for 3 h with 6-dimethylaminopuri ne (6-DMAP) following either ionomycin or ethanol activation. While ov er 80% of these oocytes cleaved after transfer to the oviducts of reci pients, progression to the blastocyst stage was higher after ionomycin as compared with ethanol activation (58% vs. 19%). The ionomycin plus 6-DMAP activation protocol was used to produce parthenogenetic blasto cysts whose subsequent development was monitored both by ultrasonograp hy and by direct fetal examination. Over 70% of parthenogenotes were v iable on Day 21 of pregnancy but dead by Day 25. The effects of 6-DMAP on nuclear remodeling and fetal development of cloned embryos was the n investigated. Control cloned embryos underwent nuclear envelope brea kdown (NEBD), premature chromatin condensation (PCC), and inhibition o f DNA synthesis. By contrast, reconstructed embryos treated with 6-DMA P exhibited intact nuclear membranes, interphase chromatin, and no int erference on DNA synthesis. Moreover, cloned embryos developed to blas tocyst;stage in higher percentage after 6-DMAP treatment (83% vs. 25%) . We conclude that ionomycin followed by 6-DMAP incubation yields high percentages of diploid parthenogenetic embryos that develop to Day 25 before dying. Cloned embryos activated by the ionomycin-6-DMAP protoc ol develop readily to term.