USE OF STORED PIG EGGS TO ASSESS BOAR SPERM FERTILIZING FUNCTIONS IN-VITRO

Methodology for studying sperm-zona interaction in the pig was establi shed using cryopreserved cumulus-free immature eggs obtained in large numbers from gilts' 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 transf erred to fresh sperm-free medium so that the behavior of the egg-assoc iated sperm sample could be assessed during further incubation (''post incubation''). Complexes were passaged repeatedly through a wide-bore pipette tip to rinse off loosely bound sperm and leave tightly bound s perm; alternatively, they were passaged repeatedly through a narrow-bo re pipette tip to strip off sperm bound to the zona surface, whence re sidual zona-penetrated sperm could be counted. Zona-binding ability wa s present in the sperm populations very soon after the start of preinc ubation, although it increased during the following 3 h; considerable binding was also noted in a medium that did not support IVF. Zona-pene trating ability was absent at the start of preincubation, increased sl owly 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 complexe s. However, numbers of penetrated sperm rose slowly in a curvilinear f ashion to maximize after some 3 h of postincubation, when they constit uted less than about 15% of the bound sperm. No rapidly penetrating co hort was detectable, and the proportion of sperm that became tightly b ound to the zona was unaffected by either preincubation or postincubat ion. It was concluded that 1) the strength of sperm-zona attachment re flected the area of the sperm head in contact with the zona rather tha n any physiologically specific binding and 2) zona attachment was not a functional or temporal indicator of zona penetration.