Use of stored pig eggs to assess boar sperm fertilizing functions in vitro

Methodology for studying sperm-zona interaction in the pig was established using cryopreserved cumulus-free immature eggs obtained in large numbers from giltsʹ ovaries. Boar sperm were preincubated in a medium known to support in vitro fertilization (IVF) and coincubated briefly with groups of eggs, and the resultant sperm-egg complexes were transferred to fresh sperm-free medium so that the behavior of the egg- associated sperm sample could be assessed during further incubation ("postincubation"). Complexes were passaged repeatedly through a wide- bore pipette tip to rinse off loosely bound sperm and leave tightly bound sperm; alternatively, they were passaged repeatedly through a narrow-bore pipette tip to strip off sperm bound to the zona surface, whence residual zonapenetrated sperm could be counted. Zona-binding ability was present in the sperm populations very soon after the start of preincubation, although it increased during the following 3 h; considerable binding was also noted in a medium that did not support IVF. Zona-penetrating ability was absent at the start of preincubation, increased slowly to a maximum after 3 h, and declined thereafter; penetration was insignificant in the medium that did not support IVF. Associated sperm numbers remained constant during postincubation of sperm-egg complexes. However, numbers of penetrated sperm rose slowly in a curvilinear fashion to maximize after some 3 h of postincubation, when they constituted less than about 15% of the bound sperm. No rapidly penetrating cohort was detectable, and the proportion of sperm that became tightly bound to the zona was unaffected by either preincubation or postincubation. It was concluded that 1) the strength of sperm-zona attachment reflected the area of the sperm head in contact with the zona rather than any physiologically specific binding and 2) zona attachment was not a functional or temporal indicator of zona penetration.