138 TIME-LAPSE CINEMATOGRAPHY-COMPATIBLE INJECTION-MOLDED MICROWELL CULTURE SYSTEM FOR TRACKING THE DEVELOPMENT OF INDIVIDUAL BOVINE EMBRYOS

Abstract

We have developed a polystyrene-based well of-the-well system (WOW) using injection moulding to track individual embryos throughout culture using time-lapse cinematography (TLC). The WOW-cultured bovine embryos following in vitro fertilization (IVF) were compared with conventional droplet (control)-cultured embryos on in vitro and in vivo development. Twenty-five of zygotes were cultured in each culture system containing 125 μL of CR1aa medium supplemented with 5% calf serum for 168 h after IVF. No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality, inner cell mass (ICM), and trophectoderm (TE) cell numbers and post-vitrification survival rates were not different between control- and WOW-derived blastocysts; however, incidence of apoptosis in the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at days 30 and 60 after embryo transfer (P < 0.05). The TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pick-up; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of 2 blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.