Figure 1.

Incubation of sperm with ouabain or anti-ATP1A4 immunoserum-capacitated fresh bovine sperm. In vitro matured cumulus dissociated bovine oocytes were coincubated with fresh bovine sperm capacitated by incubating under various conditions (A: Sp-TALP alone; B: ouabain; C: anti-ATP1A4) for 6 hr. The oocytes were removed from the fertilization medium, washed 7 times by pipetting to remove loosely attached spermatozoa, and then mounted on a poly-L-lysine coated microscopic glass slide. The oocytes were fixed in a solution of glacial acetic acid and absolute ethyl alcohol (1:3 ratio) for 24–36 hr and stained by placing in small drops of aceto-orcein. The number of sperm bound to the zona pellucida (B,C: arrow heads indicate sperm bound to the zona pellucida) was counted by phase-contrast microspcopy. This experiment was done in three replicates, using ejaculates from three bulls. To determine the ability of sperm capacitated under above conditions to penetrate oocytes and development into pronuclei (D,E: arrow heads indicate pronuclei), in vitro matured COC were fertilized with sperm incubated in different capacitating conditions (D: anti-ATP1A4; E: ouabain). After 6 hr of sperm–oocyte coincubation, COC were cumulus-dissociated and cultured for an additional 12 hr in SOF medium. Presumptive zygotes were fixed, stained and examined microscopically for pronuclei formation at 400× magnification. This experiment was done in three replicates, using semen from different bulls.