Numbers of sperm associated with the perivitelline layer of seabird (and other bird species) eggs

Following copulation, females of many seabird species spend a prolonged period of time away from the colony, building up reserves for egg formation and incubation. Here, we report that the number of sperm associated with eggs of single-egg clutch seabirds was almost an order of magnitude greater than predicted from the relationship between ovum size and sperm numbers in multi-egg clutch non-seabirds. Sperm numbers were also several times greater than the estimated number necessary for maximal fertilisation success. Our results are consistent with three unusual features of seabird reproduction: (1) single egg clutches, (2) prolonged sperm storage, and (3) a lag period between the end of yolk formation and ovulation. We hypothesise that sperm release from storage is under precise temporal control in these species, with high sperm numbers acting as an insurance against infertility in single-egg clutches. If true, the lag period may have evolved to provide sufficient time for sperm to be released simultaneously from storage and accumulate at the site of fertilization prior to ovulation.

Methods
We obtained eggs of six single-egg clutch seabird species, from two unrelated taxa, the Alcids (Common Guillemot Uria aalge (N = 5), Puffin Fratercula arctica (N = 2), and Razorbill Alca torda (N = 5)), and the Procellariiformes (Manx Shearwater Puffinus puffinus (N = 3), Northern Fulmar Fulmarus glacialis (N = 2), and Storm Petrel Hydrobates pelagicus (N = 6). All eggs were collected under license (from Natural England, Natural Resources Wales, Scottish Natural Heritage, and the Faeroese Museum of Natural History), on the day they were laid, from Skomer Island, Wales (Guillemot, Manx shearwater, Puffin, and Razorbill), the Faroe Islands, Denmark (Storm Petrel), and Fair Isle, Scotland (Fulmar). Eggs were opened and the yolk was fixed in 5% formalin. Once fixed, the yolk diameter was measured with digital vernier calipers (0.01mm) to estimate mean ovum surface area for each species. Two 0.5 cm2 pieces of perivitelline layer per egg – one from above the germinal disc and the other from the vegetal pole – were removed from the yolk, cleaned in phosphate buffered saline (PBS) solution, and mounted on a microscope slide for examination. 10µl of the fluorescent DNA stain Hoechst 33342 (0.05mg/ml) was added to each perivitelline layer sample to stain sperm nuclei, as described by Birkhead et al. (2008).  The number of sperm trapped in and holes made by sperm in the perivitelline layer and was counted for each perivitelline sample, under a fluorescence microscope with a BP 340-380 excitation filter and a LP 425 suppression filter, dark-field optics, and x 20 objective lens. Our methods for counting sperm and holes were identical to those used by Birkhead et al. (1994), except that we did not correct for a “halo” of holes in the germinal disc region, as we found no evidence for this phenomenon in our samples. Instead, we found that the number of sperm and holes did not differ significantly between the germinal disc and vegetal pole (sperm: t = -0.01, d.f. = 22, p = 0.996; holes: t = 1.51, d.f. = 22, p = 0.145), so we calculated the mean sperm and hole number per unit area of perivitelline layer, and from this estimated the total number of sperm and holes for each ovum.