Influence of calcium ionophore on the fertilization of bovine oocytes and their further embryonic development

Abstract

Intracytoplasmic spermatozoid injection (ICSI) is one of the commonest methods used in assisted reproductive technologies in human medicine. However, this procedure has low efficacy for bovines, mainly because of insufficient activation of oocytes after spermatozoid microinjection. One of the most effective methods of activating oocytes is considered to be the use of phosphorus calcium, though the optimal concentration of activator and its effect on pre-implant development of embyo are still open questions. An oocyte-cumulus complex of clinically healthy cows, retrieved from the ovaries during slaughter, matured over 22–24 h in in vitro conditions. Oocytes with visible polar body had been subjected to intracytoplasmic spermatozoid injection (ICSI), and were 15–30 min later activated in the environment with different concentrations of calcium ionophore for 15–20 min and then transferred for further cultivation in a culture medium with sodium pyruvate. The fertilization rate was identified on the second day at the 2–4th stages of cellular embryo, and the quality of obtained embyos was evaluated on day 8. Based on the statistical analysis of the data, we determined that the artificial activation of bovine oocytes using calcium ionphore after intracytoplasmic spermatozoid injection (ICSI) led to statistically significant improvement in conception and ratio of blastocytes obtained to oocytes injected. In the study, we confirmed that addition of 5, 10 and 50 µМ of the agent had the same efficacy on the activation of occytes of bovine cattle. However, it has to be noted that during further cultivation of the obtained zygotes up to the blastocyte stage (day 8), we saw no significant differences in quality of embryos obtained. Therefore, use of calcium ionophore for the activation of bovine oocytes after intracytoplasmic spermatozoid injection is effective, for it promotes increase in fertilization parameters and ratio of blastocytes obtained to oocytes injected, facilitating production of higher numbers of embyos suitable for transplantation or cooling. Our previous conclusions are valuable for increasing the efficacy of methods of intracytoplasmic injection of bovine spermatozoid and its further use for purposes of science and production.