Effect of heparin on the in vitro fertilizing ability of goat spermatozoa as assessed by heterologous solubilized zona pellucidae

Abstract

Heparin has been shown to improve the fertilizing ability of goat spermatozoa in a dose-dependent manner. The present study assessed the mechanism involved in the stimulatory effect by using solubilized zona pellucidae from bovines.

Bovine cumulus-oocyte complexes were matured in 199 plus serum and insuline. Thereafter, oocytes were denuded from granulosa cells and used either to obtain zona pellucidae or for fertilization. Free zonae were collected by mechanical means, solubilized in heated acetic acid, liofilized and stored at -20° C until their reconstitution in Talp plus serum.

In Experiment 1, spermatozoa were suspended either in non-capacitated conditions or in Talp plus heparin and Talp plus heparin and zonae, and incubated for 90 min at 39° C in 5% CO2 in air. In Experiment 2, spermatozoa were grouped as before and co-incubated with matured oocytes for 18 hr to assess their fertilizing ability. In Experiment 3, spermatozoa were suspended either in seminal plasma, Talp plus heparin or Talp without heparin for 90 min. At 0, 45 and 90 min, semen samples were exposed to solubilized zonae and were further incubated for 30 min. Acrosome disruption in Experiment 1 and 3 was assessed by staining spermatozoa with PI/PSA. Fertilization in Experiment 2 was verified by the presence of pronuclei and sperm tail. 
The results in Experiment 1 showed that the acrosome disruption rate increased above the control groups after 90 min of incubation only in zonae-treated spermatozoa (P<0.001). In Experiment 2, fertilization was seen only when spermatozoa were previously incubated in conditions known to capacitate them. In Experiment 3, the results showed that the acrosome disruption rate first increased when spermatozoa were exposed to zonae after 45 min of incubation (P<0.01).

The cumulative results suggest that heparin improves fertilization by stimulating sperm capacitation. The effect is expressed after 45 min of sperm incubation.