When used as a solo maturation medium for oocytes, porcine follicular fluid (pFF) promoted male pronucleus formation (MPF) of oocytes after in vitro maturation (IVM), using a static system, and in vitro fertilization (IVF) in pigs (Naito et al. 1988 Gamete Res. 21, 289–295). However, the developmental competence of oocytes matured in pFF after IVM/IVF has not been reported. This study was conducted to assess the ability of pFF as a maturation medium to support IVM/IVF of porcine oocytes and their subsequent in vitro development. pFF, including cumulus–oocyte complexes (COCs), was aspirated from follicles (2–5 mm in diameter) of prepubertal crossbred gilt ovaries, and large clusters of follicular cells (FC) were removed from pFF by filtration through 212 �m of mesh. All of the COCs in filtered pFF were collected, and COCs with compact cumulus cells were selected for IVM. Also, small clusters of FC were collected by centrifugation of the filtered pFF, and pFF without any cells was prepared by centrifugation and used as a maturation medium (MpFF) after supplementation with FSH and antibiotics. COCs were transferred to 3.5 mL (in a 15-mL test tube) of MpFF with FC (5.2 � 106 cells mL-1) and cultured for 44–48 h at 38.5�C in 5% O2 and 5% CO2 using the rotating culture system. As a control group, COCs were cultured in 2 mL of MpFF without FC in a 35-mm Petri dish by the standard static culture system. After maturation, culture oocytes were co-incubated (IVF) for 5 h with frozen–thawed sperm in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041), and then some of them were fixed 10 h after IVF to assess the fertilization status; the rest of them were cultured in PZM (Yoshioka et al. 2002 Biol. Reprod. 60, 112–119) for 7 days to assess their early embryonic development. All of the data were analyzed by ANOVA. Oocytes cultured with FC in the rotating system (R group) showed significantly higher sperm penetration (71.0%), MPF formation (70.5%), and normal fertilization (monospermic fertilization with female and male pronuclei; 31.5%) rates than those in the control group (56.0%, 56.9%, and 17.6%, respectively; P < 0.05). Also, the R group showed significantly higher rates of 8-cell embryos at 2 days after IVF and blastocyst formation at 7 days after IVF than those of the control group (17.2% vs. 8.3% and 10.9% vs. 4.5%, respectively; P < 0.05). These results indicate that porcine oocytes matured in pFF supplemented with FC using the rotating system have the ability to be penetrated by sperm and form MPF, and to develop to the blastocyst stage at higher rates, than oocytes cultured in the standard static maturation culture system. In conclusion, the pFF can be a sole and simple maturation culture medium useful for the in vitro production of blastocysts in pigs.
In vitro maturation of ovine oocyte in a modified granulosa cells co-culture system and alpha-tocopherol supplementation: effects on nuclear maturation and cleavage
