Fertilization of pig oocytes was performed in vitro in modified Tyrode's media in which either HEPES or bicarbonate/CO2, or both, were included as buffer systems; caffeine (2 mM) was also included in some of the media because it is a reported stimulant of fertilization. The composition of the bicarbonate-containing media was designed so as to maintain the same pH and osmolality as bicarbonate-free media. The inclusion of bicarbonate during gamete co-incubation in caffeine-containing medium led to high levels of fertilization (66% of 238 mature oocytes were fertilized). However, essentially no fertilization occurred if bicarbonate was replaced with HEPES (0.7% of 146 oocytes were fertilized; significantly different, P < 0.001). Inclusion of HEPES in bicarbonate-containing medium during gamete co-incubation did not affect fertilization, showing that HEPES did not exert an inhibitory effect. Omission of bicarbonate during sperm preincubation also did not affect fertilization. If caffeine was included in bicarbonate-containing medium, 73% of 311 oocytes were fertilized whereas if caffeine was omitted only 14% of 326 oocytes were fertilized (significantly different, P < 0.001). In the absence of bicarbonate, when fertilization was very low, caffeine had no stimulatory effect. The results indicate that bicarbonate is essential for pig fertilization in vitro, but that caffeine exerts a synergistic stimulatory effect.

The present study was conducted to investigate the direct action of hypoxanthine, which has been reported to be present in pig follicular fluid, on spontaneous meiotic resumption of pig denuded oocytes in vitro. No oocytes, which were surrounded by a cumulus and some membrana granulosa cells directly adjacent to it from healthy antral follicles, underwent germinal vesicle breakdown (GVBD) when they were cultured in serum-supplemented 199 with or without 4 mM hypoxanthine. On the other hand, 81% of denuded oocytes prepared from such cumulus-oocyte-granulosa cell complexes showed GVBD after 24 h of culture. Hypoxanthine inhibited significantly this spontaneous resumption of meiosis in denuded oocytes. The inhibitory action of hypoxanthine was dose dependent at concentrations of 1-6 mM and was reversible following subsequent culture of the oocytes in hypoxanthine-free medium. However, premature chromosome condensation was observed in oocytes cultured in hypoxanthine-supplemented medium, and the oocytes released from the influence of hypoxanthine completed GVBD and reached the second metaphase slightly faster than those freshly isolated from the follicle. These results suggest that hypoxanthine inhibits spontaneous meiotic resumption of pig oocytes in vitro. However, the inhibitory action of hypoxanthine does not prevent all events involved in GVBD from occurring.

In Exp. 1 oocytes were collected from follicles (4-6 mm) in ovaries from prepubertal gilts by aspiration of the follicles with a syringe or dissection from healthy follicles. After aspiration some oocytes with compact cumulus and granulosa cells were selected. In Exp.2 oocytes were collected from follicles (4 mm, 5-6 mm and 7-8 mm) of gilts on the 18th-19th day of the cycle. Germinal vesicle (GV) stages of oocytes were examined and categorized into GV1, GV2, GV3 and GV4. In Exp. 1 when collected by aspiration, a significantly lower percentage of oocytes (45%) was arrested at the GV1 stage than oocytes from dissected follicles (78%). However, after morphological selection of oocytes, the percentage (58%) significantly increased. In Exp 2, significantly higher percentages (86-90%) of oocytes from follicles of 5-8 mm in diameter were at the GV1 stage than oocytes from follicles in diameter of 4 mm (24%) and atretic follicles (57%). These results indicate that by aspiration some oocytes are collected from follicles that were inadequate to maintain arrest at GV1, but significantly higher percentages of oocytes at GV1 can be obtained after morphological selection of oocytes or isolation from large follicles of gilts on the 18th-19th day of the cycle.

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