306 IN VITRO PENETRATION OF SWINE OOCYTES MATURED IN TCM-199 WITH ADDED DIBUTYRYL CYCLIC ADENOSINE MONOPHOSPHATE AND FOLLICULAR FLUID

Abstract

During the in vitro maturation (IVM) of pig oocytes, a large variation in the nuclear morphology of the germinal vesicle stage is observed. Thus, some oocytes can start meiosis earlier than others. A reversible alternative to inhibit meiotic resumption is the use of dibutyryl cyclic adenosine monophosphate (dbcAMP) in the early period of IVM, which may synchronize the oocytes to a specific germinal vesicle stage and improve early embryonic development. This study investigated the effects of additional dbcAMP and porcine follicular fluid (PFF) on monospermic and polyspermic penetration rates after IVF. Oocytes from prepuberal females were selected under a stereomicroscope, and those with uniform ooplasm and surrounded by several layers of compact cumulus cells were divided into 2 groups: T1 (control) group: TCM-199 supplemented with polyvinyl alcohol (0.1%), FSH (0.5 �g mL-1), LH (0.5 �g mL-1), epidermal growth factor (10 ng mL-1), pyruvate (0.9 mM), d-glucose (3.05 mM), cysteine (0.1 mg mL-1), and gentamycin (50 �g mL-1); and T2 group: T1 with the addition of 25% PFF and 1 mM dbcAMP. Both the T1 and T2 groups were IVM for 42 to 46 h, with FSH, LH, and dbcAMP used only in the first 22 h. At the end of the maturation period, cumulus cells were chemically removed; the oocytes were washed 3 times in IVF medium (modified Tyrode's buffered medium, mTBM) and placed in petri dishes containing 50 µL of the same medium. The sperm-rich fraction was collected from 2 boars by digital pressure with a gloved hand, extended in Beltsville thawing solution, and incubated for 24 h at 17°C. It was then centrifuged at 1200g for 3 min and standardized for 1 × 105 spermatozoa mL−1. Oocytes were co-incubated with the sperm for 6 h in mTBM at 38.5°C and 5% CO2. After insemination, oocytes were cultured in porcine zygote medium-3 (80 µL), covered with paraffin oil, for 18 h. The presumptive zygotes were fixed and stained with 1% orcein in 45% acetic acid and evaluated under phase-contrast microscopy at a 400× magnification. Differences among groups were determined by one-way ANOVA. In the T1 group, the penetration rate was 39.3 ± 9.6% (162/412), and no difference was observed in comparison with the T2 group, 29.5 ± 4.9% (113/383). The monospermic penetration rate was 31.5 ± 6% (51/162) in the T1 group and differed from that in the T2 group, 71.7 ± 3.3% (81/113). Moreover, the polyspermic penetration was significantly higher in the T1 group, 68.5 ± 6% (111/162), compared with the T2 group, 28.3 ± 3.3% (32/113). These data suggest that the IVM with TCM-199 with added dbcAMP + PFF can improve in vitro production of swine embryos and decrease polyspermic penetration.