A continuous flow, perifusion culture system for 8- to 16-cell bovine embryos derived from in vitro culture

Eight- to 16-cell stage bovine embryos derived from in vitro maturation and in vitro fertilization were obtained after static culture until 80 to 88 h post insemination. The embryos were cultured in a dynamic (continuous flow) perifusion culture system for 24 h and were further co-cultured on a cumulus cell monolayer in a static CO2 incubator for an additional 80 to 88 h. Based on preliminary experiments, a serum-free bovine embryo culture medium (BECM) supplemented with 25 mM HEPES was used as the perifusion culture medium (PCM), and the flow rate was fixed within the range of 30 to 38 microliter/min in subsequent experiments. In Experiment 1, the combined effects of tubing (silicone vs Tygon) and/or culture chamber size (0.2 vs 1.5 ml) on prehatching development of the embryos following 24 h perifusion culture were evaluated in an experiment with a 2 X 2 factorial arrangement. More (P < 0.005) embryos developed to the blastocyst stage with Tygon than with silicone tubing, regardless of chamber size. A higher percentage of blastocysts (29%) was obtained following the perifusion culture by the combination of Tygon tubing and the 1.5-ml culture chamber than by any other combination. In addition, a significantly (P < 0.001) lower mean pH of the perifusate was obtained with Tygon (7.57 to 7.58) than with silicone (7.78 to 7.79) tubing, regardless of chamber size. In Experiment 2, embryo development was compared after culture in the perifusion system and in a static culture system using either serum-free medium (control) or serum-free medium plus a cumulus cell monolayer (co-culture). Percentages of morulae (49 vs 57%) and blastocysts (31 vs 33%) obtained from the control system were not significantly (P > 0.4) higher than the percentages obtained from the perifusion system. However, these proportions were significantly (P < 0.01) increased by co-culturing the embryos on a cumulus cell monolayer in the static incubator for the same intervals (71% morulae and 44% in blastocysts). In conclusion, 8- to 16-cell bovine embryos can develop to the blastocyst stage following 24 h perifusion in the dynamic culture system using a serum-free medium. Physical stress due to flow rate within the range of 30 to 38 microliter/min in an air atmosphere has little effect on the development of perifused embryos cultured under this system.