213 POLARIZED LIGHT MICROSCOPY REVEALS AN ASSOCIATION BETWEEN ZONA BIREFRINGENCE INTENSITY AND EMBRYONIC DEVELOPMENT IN BOVINE OOCYTES AND ZYGOTES

Abstract

To increase the efficiency of human IVP, noninvasive parameters to predict the developmental competence of oocytes and zygotes would be useful for selecting the right embryo to transfer. However, human oocytes and zygotes for experimental studies are rare. Therefore, using a bovine model with a precisely large sample size was the aim of the present study to investigate whether zona birefringence intensity (ZBI), measured by polarization light microscopy of metaphase II (MII) oocytes and zygote-stage embryos, was associated with subsequent development. In this study, ZBI was measured through 2 different parameters, the birefringence peak (PV, average signal strength of polarized light) and the birefringence peak combined with the surface of the signal (CV, average signal strength of polarized light multiplied by the surface of the signal), by using polarized light microscopy and OCTAX polarAIDE-software. Statistical analysis was done by the Tukey-Kramer test using SAS version 9.1. Cumulus–oocyte complexes (COC) were recovered from slaughterhouse ovaries by aspiration. After in vitro maturation, MII oocytes were denuded and activated parthenogenetically (4-min exposure to 5 μm Ionomycin, followed by 3.5 h of incubation in 2 mm 6-DMAP) or fertilized in vitro before denudation. Subsequently, ZBI of MII oocytes (directly after activation) and zygotes (24 h after the beginning of IVF) were measured. To allow tracking of subsequent development, embryos were cultured individually in a well-of-well (WoW) culture system until Day 7 in CR1aa medium (5% CO₂, 38.7°C, humidified air). When parthenogenetically activated embryos were cultured (n = 365), 287 (78.6%) cleaved and 115 (31.5%) reached blastocyst stage. The ZBI of MII-activated oocytes did not indicate any differences between those that cleaved and those that did not. On the contrary, we observed significant differences (P < 0.05) for mean values of PV (41.4 v. 43.3) and CV (21.3 v. 22.6) for MII-activated oocytes that subsequently reached the blastocyst stage at Day 7 and those that did not. When IVF zygotes (n = 415, 80.5% cleavage rate and 26.0% blastocyst rate) were analyzed, significantly (P < 0.05) lower mean values of PV (47.1 v. 51.5) and CV (24.7 v. 27.1) were observed for zygotes that cleaved within 48 h compared with zygotes that did not cleave. In addition, mean values of PV (45.5 v. 47.9) and CV (24.3 v. 25.4) for the zygotes that developed to the blastocyst stage at Day 7 were significantly lower in comparison with zygotes that did not develop to that developmental stage. In conclusion, we observed a connection between birefringence intensity at the MII/zygote stage and further development. This may offer benefits for the identification of favorable MII oocytes that could have implications for bovine somatic cell nuclear transfer as well as for human-assisted reproduction technology.