Assessment of bovine oocytes quality and their resistance to age-related changes after maturation in single-phase and two-phase culture systems

Abstract

Existing approaches to in vitro maturation (IVM) of bovine oocytes do not take into account their specific demands during terminal phase of IVM including the need for increasing of their resistance to age-re-lated changes. In this work, we performed for the first time a comparative investigation of nuclear maturation and abnormal changes of MII chromosomes in bovine oocytes after their maturation in single-phase system and different two-phase systems and after the subsequent prolonged culture of the ova. When using the single-phase system, cumulus-oocyte complexes (COCs) were cultured for 24 h in the medium TCM 199 containing 10% fetal bovine serum (FBS), 10 μg/ml FSH, and 10 μg/ml luteinizing hormone (LH). In the two-phase system, oocytes matured in the same conditions for first 16 h and then in a new medium (TCM 199 containing 10% FBS (Control) or the same medium supplemented with 50 ng/ml progesterone) for the remaining 8 h of IVM. After maturation in the single-phase and the two-phase systems, a part of COCs were transferred to an aging medium (TCM 199 containing 10% FBS) and further cultured for 24 h. The state of the nuclear material (stage of meiosis and abnormal changes of MII chromosomes) in the matured and aged oocytes was assessed using cytogenetic analysis. The rate of oocytes being at the MII stage of meiosis after IVM in the single-phase and the two-phase systems was similar and amounted 82.7-86.3%. In addition, no effect of the culture system on the rate of MII oocytes with abnormal morphology of chromosomes was revealed. For the single-phase culture, this rate after the end of the IVM period was 32.2±0.5% and for the two-phase systems culture 38.5±4.0%. The prolonged culture of matured oocytes led to an increase the frequency of destructive changes in MII chromosomes to 56.9±2.9 and 68.4±3.0%, respectively. Progesterone in the IVM medium (during the last 8 hours) also did not effect on the rate of nuclear maturation, but reduced the rate of oocytes with abnormal changes in chromosomes: after IVM, as compared with the Control and the two-phase system; after 24-hours prolonged culture as compared to control (P <0.05). The results of the study suggest that the two-phase maturation of bovine oocytes may be used as an alternative to the conventional IVM protocol and that progesterone during the second phase of IVM causes an increase in the quality of ova and their resistance to subsequent age-related transformations.