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Regulation of in vitro penetration of frozen–thawed boar spermatozoa by caffeine and adenosine

Effects of caffeine and adenosine on the function and in vitro penetration of frozen–thawed boar spermatozoa were examined. First, the effect on sperm function was determined by the chlortetracycline fluorescence assessment. Both caffeine and adenosine stimulated capacitation of spermatozoa. However, adenosine, but not caffeine, inhibited spontaneous acrosome loss. Second, sperm penetration into in vitro matured oocytes was compared among spermatozoa cultured in the absence or presence of caffeine or adenosine. Both caffeine and adenosine increased the penetration rate (99.1 ± 0.9% in caffeine, 72.4 ± 2.0% in adenosine vs. 54.8 ± 5.1% in controls) but only caffeine decreased drastically the monospermic penetration rate (8.0 ± 2.3% in caffeine vs. 75.4 ± 4.8% in adenosine and 78.6 ± 4.8% in controls). When oocytes were cocultured in various sperm concentrations, the proportion of monospermy changed in inverse proportion to sperm concentration in the presence of caffeine, but did not change in the presence of adenosine. A relatively high number of spermatozoa at the early stage of spontaneous acrosome reaction in the presence of caffeine may be one of the main causes of polyspermic penetration in porcine IVF system. These results indicate that replacement of caffeine with adenosine in fertilization medium improves monospermic penetration by frozen–thawed boar spermatozoa. Mol. Reprod. Dev. 58:424–431, 2001. © 2001 Wiley‐Liss, Inc.