154 THE EFFECT OF OXYGEN TENSION ON IN VITRO DEVELOPMENT OF BOVINE EMBRYOS IN POLYDIMETHYLSILOXANE-BASED WELL OF THE WELL DISHES PREPARED UNDER ATMOSPHERIC OR REDUCED AIR PRESSURE

Abstract

Polydimethylsiloxane (PDMS) is a non-toxic silicon compound. Its excellent optical characteristics and easy preparation make it a good candidate material for the molding of custom-shaped dishes for embryo culture. We investigated the feasibility of PDMS-based well of the well (WOW) dishes for in vitro culture of bovine embryos under different oxygen tensions. The WOW dishes with 25 micro-wells (each of 175 μm depth and 250 μm width in diameter arranged in 5 columns and 5 rows) were molded from PDMS prepared either under atmospheric (Experiment 1) or reduced (0.1 MPa) (Experiment 2) air pressure to remove air bubbles. Presumptive zygotes obtained by the in vitro maturation and fertilization of follicular oocytes were placed and cultured for 7 days in traditional micro-drops of culture medium (Control) or in the micro-wells of PDMS-based WOW dishes (PDMS-WOW), both covered by paraffin oil. The culture medium was CR1aa supplemented with 5% calf serum. The culture drop size was 125 μL (5 μL/oocyte) in both groups. Embryo development and blastocyst cell numbers between Control and PDMS-WOW groups were compared either under 20% or 5% O2 tensions. There was no statistical difference in cleavage and blastocyst rates (ranging between 82.3-86.4% and 34.0-45.8%, respectively) between Control and PDMS-WOW embryos irrespective of oxygen tension and dish production method. In Experiment 1, the mean total cell numbers in blastocysts were lower in the PDMS-WOW group than that in Control under 20% O2 (105.0 ± 5.5 and 130.4 ± 9.9, respectively) (P < 0.05, ANOVA); however, the application of 5% O2 significantly improved the cell numbers and eliminated the difference between the PDMS-WOW and Control groups (135.4 ± 6.2 and 148.0 ± 9.0, respectively). In Experiment 2, there was no significant difference in mean total cell numbers in blastocysts between the PDMS-WOW and Control either under 20% O2 (97.2 ± 5.7 and 103.9 ± 8.9, respectively) or 5% O2 (147.5 ± 12.1 and 157.3 ± 3.9, respectively). The numbers and rates of inner cell mass and trophectoderm cells did not differ between the Control and PDMS-WOW groups, irrespective of O2 tension and production method. Our results demonstrate that bovine embryos can develop to the blastocyst stage in PDMS-based WOW dishes; however, it may express detrimental effects on embryonic cell numbers, which can be neutralized by the application of low O2 tension during culture or reduced air pressure during the PDMS preparation. This work was supported by the Research and Development Program for New Bio-Industry Initiatives.