Individual serum-free and oil-based oocyte-to-embryo in vitro culture system is yielding high blastocyst rates and can be used as a basic system for individual follow-up

Bovine in vitro embryo production (IVP) is routinely performed by culturing oocytes in group at ratio 1:2 (25/50µL droplets). We have recently shown that individual culture of bovine embryos in SOF-medium supplemented with 0.4 % BSA and insulin, transferrin and selenium (SOF-ITS-BSA) is yielding day 8- blastocyst rates over 40 %. However, in order to get these high blastocyst rates, in vitro maturation and fertilization still have to be performed in group culture. Several groups have attempted to develop an in vitro maturation-fertilization-culture system allowing individual follow-up from oocyte until embryo. Different approaches such as attaching the oocytes to the bottom of the Petri dish with Cell-Tak®, using a mesh grid or culturing oocytes and embryos in the well-of-the-well system have been attempted. These systems work well but are technically often challenging. Here we describe a simple individual oocyte-toembryo culture system which is yielding routinely over 30 % blastocyst rates. In vitro maturation, fertilization and culture were either performed in group or in individual culture. For group culture, sixty cumulus-oocyte complexes were aspirated from ovaries derived from cows slaughtered in a local abattoir and matured in 500 µL TCM199 supplemented with 20 ng/mL EGF for 22h. Next, mature oocytes were incubated in 500 µL IVF-TALP with 1 × 106 spermatozoa/mL for 20h and then denuded and cultured in groups of 25 presumed zygotes in 50 µL droplets of SOF-ITS-BSA under paraffin oil (7.5 mL ) overlay. For individual culture, 3 dishes (60×15 mm) with 20 µL droplets under paraffin oil overlay were used, each droplet containing one cumulus-oocyte-complex for maturation and subsequent fertilization (in the same media as described for group culture), and after denudation, presumed zygotes were cultured individually in 20 µL droplets SOF-ITS –BSA under paraffin oil overlay until day 8. Each dish contained 17 droplets. Blastocysts were then subjected to differential staining. Blastocyst rates (5 replicates) were significantly lower for individual compared to group culture (32 % (79/244) versus 47 % (146/314)) (Independent sample t-test, SPSS 20; P˂0.05), but higher than 30 % so still acceptable. Blastocyst quality was also significantly lower, with a lower total cell number (90 ± 1.31 vs. 118 ± 1.16) and higher apoptotic cell ratio (8.4 ± 0.25% vs. 5.2 ± 0.19%) for individual versus group culture respectively. This indicates that despite the high overall blastocyst rates, there is still room for improvement in the individual culture system. In conclusion, the serum-free and oil-containing individual culture system we describe here is yielding acceptable blastocyst rates and can as such be used as to investigate (1) how differences in initial oocyte quality can affect embryo outcome; and (2) how addition of specific biochemical factors to the single oocyte maturation medium can be used in order to improve oocyte maturation. We are now testing the addition of different components derived from bovine follicular fluid to maturation medium in order to evaluate their possible effect on individual oocyte maturation and further embryo development.