79 EFFECTS OF EPIDERMAL GROWTH FACTOR, β-MERCAPTOETHANOL, GLUCOSE, OXYGEN CONCENTRATION, AND FIBROBLAST SUBCULTURE ON THE DEVELOPMENT OF PORCINE NT EMBRYOS

The purpose of this study was to investigate in vitro maturation rate of oocytes cultured in maturation medium supplemented with epidermal growth factor (EGF), β-mercaptoethanol (ME), and glucose, and the further development of NT embryos under various conditions. The basic media used for oocyte maturation were NCSU-23 and PZM-3 supplemented with 0.1 mg mL-1 cysteine, 10% (v/v) porcine follicular fluid (pFF), 10 �g mL-1 FSH, 10 �g mL-1 LH, 20 ng mL-1 EGF, and 25 �M ME. Porcine ovaries were collected at a local slaughterhouse, and donor cells from a 35-day-old fetus were dissociated, resuspended, and cultured for 6–8 days in DMEM supplemented with 10% (v/v) FBS, penicillin G (75 �g mL-1), streptomycin (50 �g mL-1), 1 mM sodium pyruvate, and 1% (v/v) nonessential amino acids. The first polar body and adjacent cytoplasm were enucleated by a micropipette in HEPES-buffered NCSU-23 supplemented with 4 mg mL-1 BSA and 7.5 �g mL-1 cytochalasin B. Couplets were equilibrated with 0.3 M mannitol solution and transferred to a chamber containing 2 electrodes with a pulse of 2.1 kV cm-1 for 30 �s. When the embryos were cultured in NCSU-23 or PZM-3 supplemented with or without 20 ng mL-1 EGF for 144 h, the development rates to the blastocyst stage were 12.0 � 1.3%, 9.6 � 1.9%, 10.9 � 2.1%, and 9.1 � 2.3%, respectively. When the embryos were cultured in NCSU-23 or PZM-3 supplemented with or without 25 �M ME for 144 h, the rates to blastocyst stage were 9.6 � 1.7%, 7.3 � 2.3%, 11.9 � 1.8%, and 7.4 � 2.1%, respectively. The developmental rate to the blastocyst stage of NT embryos cultured in PZM-3 supplemented with ME was significantly higher than when cultured without ME supplementation (P < 0.05). When the embryos were cultured in NCSU-23 or PZM-3 supplemented with or without 1.5 mM glucose for 144 h, the rates to blastocyst stage were 9.4 � 2.2%, 6.8 � 2.7%, 10.9 � 2.4%, and 8.9 � 2.6%, respectively. The developmental rate to the blastocyst stage of NT embryos cultured in NCSU-23 and PZM-3 supplemented with glucose was higher than when cultured without glucose supplementation. When NT embryos were cultured in NUSU-23 and PZM-3 at 5% and 20% O2 concentration, the rates were 11.1 � 1.8%, 9.8 � 1.4%, 12.5 � 1.6%, and 10.9 � 1.5%, respectively. The developmental rate to the blastocyst stage of NT embryos cultured in both NCSU-23 and PZM-3 at 5% O2 concentration was higher than when cultured at 20% O2 concentration. When fetal fibroblasts were cultured in NCSU-23 and PZM-3, the fusion rate of less than 10 passages was higher than for those of 11–15 passages. In conclusion, the present study indicates that EGF and glucose have beneficial effects on the in vitro maturation of oocytes, and ME improves the developmental ability of NT embryos. Furthermore, the developmental rate in subcultured fibroblast cells was improved when reconstruction was made with less than 10 passages.