IMPORTANCE OF BICARBONATE CO2 FOR FERTILIZATION OF PIG OOCYTES IN-VITRO, AND SYNERGISM WITH CAFFEINE/

Fertilization of pig oocytes was performed in vitro in modified Tyrode 's media in which either HEPES or bicarbonate/CO2, or both, were inclu ded as buffer systems; caffeine (2 mM) was also included in some of th e media because it is a reported stimulant of fertilization. The compo sition of the bicarbonate-containing media was designed so as to maint ain the same pH and osmolality as bicarbonate-free media. The inclusio n of bicarbonate during gamete co-incubation in caffeine-containing me dium led to high levels of fertilization (66% of 238 mature oocytes we re fertilized). However, essentially no fertilization occurred if bica rbonate 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 fertiliz ation, showing that HEPES did not exert an inhibitory effect. Omission of bicarbonate during sperm preincubation also did not affect fertili zation. If caffeine was included in bicarbonate-containing medium, 73% of 311 oocytes were fertilized whereas if caffeine was omitted only 1 4% of 326 oocytes were fertilized (significantly different, P < 0.001) . In the absence of bicarbonate, when fertilization was very low, caff eine had no stimulatory effect. The results indicate that bicarbonate is essential for pig fertilization in vitro, but that caffeine exerts a synergistic stimulatory effect.