Effect of activation protocols on in vitro development of parthenogenetic diploid bovine embryos

Abstract

This study was carried out to evaluate the efficiency of activation methods and how they effect on preimplantation development of parthenogenetic diploid bovine embryos to support some evidences for the research such as cloning animals, especially in Vietnam. The in vitro maturation oocytes were activated by various combination treatments: (a)Calcium Inophore A23187 (5μM) +6-DMAP (2mM)/CB (5μg/mL); (b)A23187 5μM+CHX (10μg/mL) /CB (5μg/mL) (c)EtOH 7%+6-DMAP (2mM)/CB (5μg/mL); (d)EtOH 7%+CHX (10 μg/mL)/CB (5μg/mL) (e)Ionomycin (5μM)+6-DMAP (2mM)/CB (5μg/mL) (f)Ionomycin (5μM)+CHX (10 μg/mL)/CB (5μg/mL) (g)Ionomycin (10μM)+6-DMAP (2mM)/CB (5μg/mL) (h)Ionomycin (10μM)+CHX (10μg/mL) /CB (5μg/mL). Generally, treatment with combination of A23187 (5μM) and CHX (10μg/mL) indicates better results comparing to others in cleavage rates, including formation of the two-cell (84.1%), the four-cell (59.1%), the eight-cell (29.5%) stage embryo (p<0.05). In contrast, the lowest rate was given by combination of EtOH 7% and 6-DMAP (2mM): 70.4% for two-cell embryos, 37% for four-cell embryos, and 11.1% for eight-cell embryos. In details, treatment (a) resulted in 81%, 50%, 27.6% following the order of two-cell, four-cell and eight-cell embryos development. Also following that order, treatment (d) gave 74.2%, 38.7%, 19.4%, treatment (e) induced 75%, 50%, 25%, treatment (f) as 80%, 57.5%, 37.5%, treatment (g) as 65%, 42.5%, 22.5%, lastly treatment (h) resulted as 71.1%, 47.4%, 23.7%. In conclusion, the results of my study suggested that the optimal activation protocol for bovine oocytes was 5μM A23187 for 5 min followed by 10μg/ml CHX for 5 hours. Keywords ✓ Bovine embryos ✓ Parthenogenesis ✓ Artificial Oocyte activation