A combined treatment of dehydroleucodine (DhL) and cytochalasin B (CB) was previously demonstrated to induce pronuclear formation of bovine oocytes (Canel and Salamone 2008 Reprod. Fertil. Dev. 21, 214-215). The aim of this study was to evaluate the potential of DhL combined with CB to induce diploid activation of parthenogenetic embryos and to employ this treatment to assist cloning by intracytoplasmic injection of whole cumulus cells. To do that, COCs were collected from cow ovaries obtained from a slaughterhouse and in vitro-matured in TCM-199, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were treated with 5 μM ionomycin (Io) for 4 min and randomly assigned to the following activation groups: a) DhL/CB (incubation with 1 μM DhL and 5 μg mL-1 CB, for 3 h); b) DhL/long CB (treatment DhL/CB for 3 h, followed by exposure to 5 μg mL-1 CB alone, for 3 additional hours); and c) DMAP (incubation with 2 mM 6-DMAP for 3 h). In experiment 1, activated oocytes underwent IVC for 48 h and cleaved embryos were treated with 1 μg mL-1 colchicine for 6 h, fixed on glass slides, and stained with 5% vol/vol Giemsa solution to assess chromosomal complements. In experiment 2, MII oocytes were mechanically enucleated and injected with whole cumulus cells obtained from IVM COCs. After 2 h, reconstructed eggs were treated with 5 μM Io for 4 min and randomly exposed to the activation treatments a, b, or c. Parthenogenetic control groups were also included. All embryos were cultured in SOF medium and rates of cleavage, morulae, and blastocysts were evaluated on Days 2, 5, and 8 (Table 1). Results showed that DhL/long CB diploidy rates were significantly higher than those of DhL/CB and DMAP (63.8, 40. and 31.6%, respectively; Fisher’s test, P < 0.05). Both DhL treatments induced polyploidy rates lower than DMAP (5.2, 10.6, and 31.6%, respectively; P < 0.05). Finally, Io followed by DhL/CB or DhL/long CB was able to induce cloned blastocyst rates not statistically different from Io plus DMAP (P > 0.05), but presumably with a higher degree of normal embryo ploidy.
Table 1.In vitro development of bovine cloned embryos activated with DhL and CB
Activation, Pronuclear Formation, and Development In Vitro of Pig Oocytes Following Intracytoplasmic Injection of Freeze-Dried Spermatozoa1
