PERIFUSION CULTURE SYSTEM FOR BOVINE EMBRYOS - IMPROVEMENT OF EMBRYO DEVELOPMENT BY USE OF BOVINE OVIDUCT EPITHELIAL-CELLS, AN ANTIOXIDANT AND POLYVINYL-ALCOHOL

Three experiments were conducted in an attempt to improve a continuous flow-perifusion system capable of maintaining embryo development for long periods of time. Bovine embryos (8-16 cells) obtained from static co-culture with cumulus cells in a serum-free medium were perifused i n an ACUSYST-S cell culture incubator. Culture chambers of the incubat or consisted of a 0.2-mL unit (Chamber 1) connected to a 1.5-mL unit ( Chamber 2), with the outflow from Chamber 1 routed to the inlet to Cha mber 2. A bovine embryo culture medium supplemented with 3 mg mL(-1) b ovine serum albumin (BSA) and 25 mM HEPES was used as a perifusion cul ture medium (PCM). Embryos were perifused in Chamber 2 for 24, 48 and 72 h and further co-cultured in a static system up to 216 h after inse mination. in Experiment 1, conditioning PCM with frozen-thawed bovine oviduct epithelial cells (BOEC) placed in Chamber 1 enhanced (P < 0.05 ) blastocyst formation of embryos in Chamber 2, after 24, 48 and 72 h of perifusion culture. The proportion of blastocysts was not further i ncreased by placing BOEC in Chamber 2 along with the embryos. In Exper iment 2, embryos were perifused with PCM conditioned with BOEC in Cham ber 1 for 48 h or 72 h. A higher proportion of perifused embryos devel oped to the blastocyst stage after addition of 25 U mL(-1) or 50 U mL( -1) of superoxide dismutase (SOD) to PCM than in its absence. However, blastocyst formation of embryos perifused for 72 h was not increased after addition of 50 U mL(-1) SOD compared with its absence. In Experi ment 3, the proportions of morulae and blastocysts were not decreased by replacement of 3 mg mL(-1) BSA with 1 mg mL(-1) polyvinyl alcohol ( PVA) in a BOEC-conditioned medium containing 50 U mL(-1) SOD after per ifusion for 48 h. In conclusion, PCM conditioning with BOEC and additi on of an antioxidant to the perifusion medium improved the development al capacity of perifused embryos. PVA is an adequate replacement for B SA in the perifusion medium.