Distribution of spermatozoa in the outer perivitelline layer from above the germinal disc of emu and ostrich eggs

In the present study, we determined the distribution of spermatozoa in the perivitelline layer above the germinal disc (GD) of emu and ostrich eggs that had been laid at random intervals after mating. Eggs were opened, the perivitelline layer overlying the GD region was collected and sperm were visualized with 4′,6′-diamidino-2-phenylindole under a fluorescence microscope. To map the distribution of sperm, the GD was divided into six areas (A–F), with A being the centre of the GD and F the area furthest from the centre. In both species, more spermatozoa were found in areas B, C and D than in areas A, E and F. More than half the GD spermatozoa were found in areas B, C and D. The pattern of distribution of spermatozoa across the GD depended on the total number of sperm in the GD. In the emu, the pattern was related to delay since last copulation and time of laying, whereas in the ostrich the pattern was related to the month of the season and the sex ratio of the mating system. When the total number of spermatozoa in the GD increased, the number of spermatozoa increased in every area of the GD, but the centre and the outer areas were the least affected. We conclude that sperm numbers are highest in a band immediately around the centre of the GD and then decline with increasing distance from the centre. The low numbers in the centre of the GD may be due to either low attractiveness of the centre for sperm or high attractiveness of the area immediately adjacent to the centre.