Activation with ionomycin followed by dehydroleucodine and cytochalasin B for the production of parthenogenetic and cloned bovine embryos.
In this work, Dehydroleucodine (DhL) was evaluated as a chemical activator of bovine oocytes and somatic cell nuclear transfer (SCNT) reconstituted embryos. Oocytes were activated with 5 microM Ionomycin (Io) and exposed for 3 h to 1 or 5 microM DhL alone (Io-Dhl1 or Io-DhL5) or combined with Cytochalasin B (Io-DhL1/CB; Io-DhL5/CB). Control groups were Io (Io), Io followed by 1.9 mM 6-Dimethylaminopurine (Io-6DMAP), and embryos produced by in vitro fertilization (IVF). Pronuclear formation and development to blastocysts of activated oocytes were evaluated. Embryos obtained by the DhL concentration that induced the highest blastocyst rates (1 microM) were karyotyped. An additional treatment based in Io-DhL1 plus lengthened (6-h) exposure to CB (Io-DhL1/long CB) was included to improve the proportion of diploid blastomeres. Finally, DhL combined with CB was employed to assist cloning by intracytoplasmic injection of whole cumulus cells. Results showed that DhL induces a pronuclear formation dynamic that was more similar to IVF-produced embryos than DMAP. Development to blastocyst stage was higher after activation with 1 microM DhL than with 5 microM DhL, either for groups combined or not with CB (19.15; 21.74 vs. 6.82; 0%, respectively) (p < 0.05). Io-DhL1 and Io-DhL1/CB treatments induced blastocyst-cleaved embryo ratios not statistically different from those of Io-DMAP (35.85%) and IVF (33.33%) groups (p > 0.05). Io-DhL1/long CB induced higher diploid blastomere rates than Io-Dhl1, Io-DhL1/CB and Io-DMAP (63.8 vs. 36.8; 40 and 31.6%, respectively) (p < 0.05). Moreover, all DhL treatments resulted in polyploidy rates that were lower than Io-DMAP (5.2, 12.0, 10.6, and 31.6%, respectively) (p < 0.05). Io-DhL1/CB and Io-DhL1/long CB induced cloned embryo blastocyst rates that were not significantly different from Io-DMAP (6.1, 9.4, and 18.3%, respectively) (p < 0.05). Our results indicate that Io-DhL1/long CB protocol could be useful for SCNT programs.
Canel
Natalia
N
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires-CONICET, Buenos Aires, Argentina.
Bevacqua
Romina
R
Fernández-Martín
Rafael
R
Salamone
Daniel F
DF
D000818:Animals; D001755:Blastocyst / Q000187:drug effects; D002417:Cattle / Q000196:embryology*; D002467:Cell Nucleus / Q000502:physiology*; D002478:Cells, Cultured; D019976:Cloning, Organism; D003571:Cytochalasin B / Q000494:pharmacology*; D047108:Embryonic Development / Q000187:drug effects*; D005260:Female; D005307:Fertilization in Vitro; D015759:Ionomycin / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D007783:Lactones / Q000494:pharmacology*; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects*; D010312:Parthenogenesis / Q000187:drug effects*; D012717:Sesquiterpenes / Q000494:pharmacology*
D007476:Ionophores; D007783:Lactones; D012717:Sesquiterpenes; C079949:dehydroleucodine; D003571:Cytochalasin B; D015759:Ionomycin