The canine oocyte penetration assay; its use as an indicator of dog spermatozoal performance in vitro

In vitro maturation and fertilisation has yet to be thoroughly investigated in the dog and is work that is required before gamete salvage programmes can be established in endangered canine species. Due to the differences which exist between the reproductive function of Canidae and other domestic species, in vitro requirements of both the oocyte and spermatozoa may also differ, and these remain to be established. The objective of this study was to investigate the ability of in vitro capacitated canine spermatozoa to penetrate the zona pellucida of in vitro matured canine oocytes. Two methods, one which utilised the fluorescent nuclear label Hoechst 33258 in combination with an aceto–orcein stain with light microscopy, and another using the fluorescence microscopy method alone were effective for staining both oocyte nuclear material and penetrated spermatozoal heads. These techniques only rarely allowed identification of both in the same oocyte. Using this assay, two Tyrodes media were compared for their ability to allow canine spermatozoa to capacitate and acrosome react in vitro as measured by the chlortetracycline and Hoechst 33258 dual fluorescence staining method. No correlation however was found between acrosomal status of spermatozoa and spermatozoal penetration of homologous oocytes, although some relationship was observed. In addition, it was found that the effect of the stage of oocyte nuclear maturation had no effect upon spermatozoal penetration and that immature oocytes could be penetrated by spermatozoa in this species.