A CONTINUOUS-FLOW, PERIFUSION CULTURE SYSTEM FOR 8-CELL TO 16-CELL BOVINE EMBRYOS DERIVED FROM IN-VITRO CULTURE

Eight- to 16-cell stage bovine embryos derived from in vitro maturatio n 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 c o-cultured on a cumulus cell monolayer in a static CO2 incubator for a n additional 80 to 88 h. Based on preliminary experiments, a serum-fre e bovine embryo culture medium (BECM) supplemented with 25 mM HEPES wa s used as the perifusion culture medium (PCM), and the flow rate was f ixed within the range of 30 to 38 mu l/min in subsequent experiments. In Experiment 1, the combined effects of tubing (silicone vs Tygon(TM) ) and/or culture chamber size (0.2 vs 1.5 ml) on prehatching developme nt of the embryos following 24 h perifusion culture were evaluated in an experiment with a 2 x 2 factorial arrangement. More (P<0.005) embry os developed to the blastocyst stage with Tygon(TM) than with silicone tubing, regardless of chamber size. A higher percentage of blastocyst s (29%) was obtained following the perifusion culture by the combinati on of Tygon(TM) tubing and the 1.5-ml culture chamber than by any othe r combination. In addition, a significantly (P<0.001) lower mean pH of the perifusate was obtained with Tygon(TM) (7.57 to 7.58) than with s ilicone (7.78 to 7.79) tubing, regardless of chamber size. In Experime nt 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-cultur e). Percentages of morulae (49 vs 57%) and blastocysts (31 vs 33%) obt ained from the control system were not significantly (P>0.4) higher th an 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 s ame intervals (71% morulae and 44% in blastocysts). In conclusion, 8- to 16-cell bovine embryos can develop to the blastocyst stage followin g 24 h perifusion in the dynamic culture system using a serum-free med ium. Physical stress due to flow rate within the range of 30 to 38 mu l/min in an air atmosphere has little effect on the development of per ifused embryos cultured under this system.