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 in an ACUSYST-S cell culture incubator. Culture chambers of the incubator 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 Chamber 2. A bovine embryo culture medium supplemented with 3 mg mL-1 bovine serum albumin (BSA) and 25 mM HEPES was used as a perifusion culture 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 insemination. 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 increased by placing BOEC in Chamber 2 along with the embryos. In Experiment 2, embryos were perifused with PCM conditioned with BOEC in Chamber 1 for 48 h or 72 h. A higher proportion of perifused embryos developed 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 Experiment 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 perifusion for 48 h. In conclusion, PCM conditioning with BOEC and addition of an antioxidant to the perifusion medium improved the developmental capacity of perifused embryos. PVA is an adequate replacement for BSA in the perifusion medium.