ESTABLISHING INDIVIDUAL BOVINE EMBRYO CULTURES IN SUB-MICROLITER ENVIRONMENT MAINTAINS THE BLASTOCYST COMPETENCE

In ART, human embryos are cultured either in groups in relatively large volumes, or individually in drops under mineral oil. However, such static culture conditions fail to mimic the physiological dynamic microenvironment experienced by human embryos in the oviduct.1,2 Microfluidic dynamic culture systems in which embryos are cultured singularly or in group in sub-microliter volumes could improve blastocyst rates and competence.3 The development of dynamic devices for single embryo culture able to monitor oxygen and other relevant metabolites through sensors is strictly dependent on the embryo ability to develop in extremely reduced media volumes. In this work, we aimed to examine the effects of the sub-microliter environment and embryo density on developmental rates and embryo quality using the bovine as animal model due to the high analogies with human embryo development. An extensive characterization of the embryo developmental rates and competence was carried out measuring blastocyst rates, blastomeres number, and apoptosis under the following culture conditions: a. conventional group embryo culture -CGEC 50/well; embryo density 1/10 μL; b. Microwell single embryo culture in 70 nL -MSEC 16/chamber; embryo density 1/70 nL; c. Microwell group embryo culture -MGEC 16/chamber; embryo density 1/3.75 μL. Our preliminary results showed that: i) individual culture in 70 nL does not perturb embryo development in comparison with MGEC; ii) a lower number of DNA fragmented blastomeres is observed in blastocysts developed in MSEC and MGEC in comparison with CGEC. The proof that confined single embryo culture in extremely reduced volumes does not impair blastocyst rates and quality is a pre-requisite for the design of microfluidic sensorized devices in which embryos are cultured and monitored singularly to identify highly competent blastocysts for single embryo transfer.