Preliminary experiments carried out on ovine oocytes were designed to establish correlations between activation protocols and subsequent rates of embryonic development. The best activation protocols were thereafter used in studies on ovine parthenogenesis and cloning. The first study established that chemical activators induce pronuclear development at a slightly higher rate than physical activation (ionomycin, 96%; ethanol, 95%; electro activation, 80%). Inhibition of second polar body extrusion and one single pronucleus were observed in the majority of the oocytes (approximately 90%) treated for 3 h with 6-dimethylaminopurine (6-DMAP) following either ionomycin or ethanol activation. While over 80% of these oocytes cleaved after transfer to the oviducts of recipients, 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 blastocysts whose subsequent development was monitored both by ultrasonography and by direct fetal examination. Over 70% of parthenogenotes were viable 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 then investigated. Control cloned embryos underwent nuclear envelope breakdown (NEBD), premature chromatin condensation (PCC), and inhibition of DNA synthesis. By contrast, reconstructed embryos treated with 6-DMAP exhibited intact nuclear membranes, interphase chromatin, and no interference on DNA synthesis. Moreover, cloned embryos developed to blastocyst 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 protocol develop readily to term.

The complete nuclear maturation and fertilization in vitro of oocytes from 30–40-day-old juvenile lambs, and their ability to develop up to the blastocyst stage when transferred into recipient ewes after fertilization in vivo and culture was studied. Cumulus–oocyte complexes were recovered from juvenile ovaries and only those with several cumulus cell layers were selected and compared with oocytes from adult sheep. The rate of meiotic progression was significantly lower (P < 0.001) for juvenile oocytes (8%) than for adult oocytes (58%) without gonadotrophins in the culture medium. However, a similar maturation rate was observed in oocytes of both juvenile (76%) and adult sheep (84%) in the presence of gonadotrophins. Eighteen hours after in vitro insemination, the fertilization rates for juvenile oocytes were not significantly different from those of adult oocytes (64% and 72%, respectively). Parthenogenetic activation and polyspermy were higher in juvenile than in adult oocytes (P < 0.001). The proportion of blastocysts produced was lower for juvenile (20% than for in vitro matured adult oocytes (49%) after their transfer into transitory recipients for 5.5 days (P < 0.01). However, the viability of blastocysts (67%) derived from in vitro matured juvenile oocytes showed a rate of hatching similar to that obtained from adult oocytes (74%). Pregnancy rates for recipient ewes at 90 days were similar for both juvenile (57%) and adult (61%) oocytes. The results indicate that it is possible to mature and fertilize in vitro matured juvenile oocytes to produce viable embryos.