146 CO-CULTURE OF EARLY CLEAVAGE STAGE IVP EMBRYOS WITH BOVINE OVIDUCT EPITHELIAL CELLS DOES NOT IMPROVE EMBRYO DEVELOPMENT OR PREGNANCY RATES

Abstract

Pregnancy rates after the transfer of bovine IVP embryos are lower than that achieved after the transfer of MOET embryos. One reason may be that the relatively defined IVC (SOFaaBSA) culture system used in vitro is suboptimal for embryo development. We investigated whether the co-culture of early stage IVP embryos with bovine oviduct epithelial cell (BOEC) to Day 3 could provide some of the missing substrates and improve both embryo production and subsequent pregnancy rates after embryo transfer. COCs were collected by ovum pickup (OPU) from donor Brahman females, transported overnight at 38.5°C to the laboratory in HEPES-IVM media, then fertilized and cultured by our standard IVP methodology (Fry et al. 2003 Theriogenology 59, 446). Briefly, the IVC was carried out at 38.5°C in a humidified atmosphere of 5% CO₂, 5% O₂, 90% N₂ in 4-well Nunc dishes in 500 μL of SOFaaBSA media overlayed by 500 μL of mineral oil. After 3 days of culture, the embryos were transferred to fresh IVC media and after 6 days placed in 5-mL Falcon tubes (Becton Dickinson Labware, Lincoln, NJ, USA) in fresh IVC media containing 2% FCS for overnight shipment. All grade 1 and 2 embryos were transferred to synchronized recipients on Day 7. Pregnancy diagnosis was between Days 50-90. In the BOEC treatment group, frozen aliquots of BOEC were thawed, seeded at 300,000 cells/mL, and grown for 2 days to 60-80% confluence in the Nunc wells in 500μL of DMEM/F12 media containing 10% FCS. On the first day of embryo culture, the media was removed and replaced by IVC media prior to the introduction of presumptive zygotes. After 3 days of co-culture, the embryos were transferred to fresh IVC media and thereafter cultured and transferred as for the Control group. In the Control group, 80 OPU sessions produced 1277 COCs (mean 16.0) of which 1064 (81.6%) cleaved producing 385 (33.7%) transferable embryos. Of the 337 embryos transferred to recipients (48 were vitrified), 141 (40.1%) resulted in pregnancies. In the BOEC group, 73 OPU sessions produced 1111 COCs (mean 15.2) of which 891 (80.2%) cleaved producing 388 (35%) transferable embryos that resulted in 161 (41.5%) pregnant recipients after transfer. Chi-square analysis showed no difference in either IVP embryo production or subsequent pregnancy rate between the Control group or the group where the IVP embryo was co-cultured for the first 3 days with BOEC.