Abstract
The Hemizona assay (HZA) was developed as a diagnostic test for predicting the fertilization potential of human spermatozoa. The aim of this study was to develop an HZA for porcine species to allow for boar semen analysis and the study of gamete interaction in this species. Porcine oocytes were obtained from slaughterhouse ovaries, denuded, and stored in buffered ammonium sulphate solution at 4 °C. On the day of the experiments the oocytes were bisected, thus providing 2 equal matching hemizonae from each oocyte. Semen samples from fertile boars were used to assess the number of spermatozoa bound to the outer side of the hemizona during incubation in vitro. The HZA index was defined as the ratio of the number of test spermatozoa bound to the hemizona divided by that of the control spermatozoa. Sperm binding to matching hemizonae of a particular boar was equal and clearly demonstrated the feasibility of using HZA for the pig. The number of spermatozoa bound to hemizona was dependent on sperm concentration in the incubation droplets. Hemizonae obtained from oocytes stored in buffered ammonium sulphate and in ammonium sulphate showed binding which was similar to that of hemizonae from fresh oocytes. However, there was a significant difference between HZA indices when fresh zonae binding was compared with hemizonae from buffered magnesium chloride-stored oocytes. A combination of monoclonal antibody 18.6 and Hoechst dye 33342 was used to assess boar sperm acrosomal status. More than 80% of the spermatozoa bound to hemizonae were partially acrosome-reacted or had lost their acrosome completely. In contrast, spermatozoa at the onset of incubation or after 4 h of incubation irrespective of exposure to hemizona showed a low incidence of acrosome reactions (14 to 25%). In conclusion, this paper presents the development and validation of an HZA for boar semen. The HZA in porcine species has several potential areas of use in which the functional status of sperm-oocyte interaction is vital.
Published Version
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