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.

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.

Below are descriptions of each data column in this dataset, separated by sheet

Sheet 1: "seabird sperm data (this study)"

common_name = species common name

species = species binomial name

spp_no_this_study = the number allocated to each species in the current study (e.g. in plots)

ovum_diameter_mm = ovum diameter in mm (note this is a mean value for each species because not all ova could be measured)

ovum_SA_mm2 = ovum surface area, extrapolated from diameter under the assumption that the ovum is spherical (4πr^2)

GD_stage = developmental stage of the germinal disc, according to the normal stages of chick development (Hamburger & Hamilton 1951)

est_development_hrs = estimated number of hours of development the embryo had undergone before egg collection, based on the germinal disc developmental stage

gd_sperm_50mm2 = the number of sperm counted in a 50mm^2 section of perivitelline layer from the germinal disc pole of the ovum

gd_holes_50mm2 = the number of holes counted in a 50mm^2 section of perivitelline layer from the germinal disc pole of the ovum

vp_sperm_50mm2 = the number of sperm counted in a 50mm^2 section of perivitelline layer from the vegetal pole of the ovum

vp_holes_50mm2 = the number of holes counted in a 50mm^2 section of perivitelline layer from the vegetal pole of the ovum

mean_total_sperm_50mm2 = the mean number of sperm and holes (summed) counted per 50mm^2 section of perivitelline layer (mean of germinal disc and vegetal pole counts)

total_sperm_ovum = the estimated total number of sperm and holes on the entire ovum, extrapolated from the mean number per 50mm^2 using the calculated ovum surface area

mean_holes_50mm2 = the mean number of holes only, counted per 50mm^2 section of perivitelline layer (mean of germinal disc and vegetal pole counts)

holes_ovum = the estimated total number of holes only on the entire ovum, extrapolated from the mean number per 50mm^2 using the calculated ovum surface area

Sheet 2: "Birkhead et al 1994 data"

common_name = species common name

species = species binomial name

spp_no_this_study = the number allocated to each species in the current study (e.g. in plots)

species_no_Birkhead1994 = the number allocated to each species in the reference study from which these data were taken, Birkhead et al. 1994 (if applicable; for two species, canada goose and mute swan, data were collected as part of the current study)

log_ovum_diameter_mm = log ovum diameter (mm) taken from Birkhead et al 1994 (read directly from plots), or measured in the current study in the case of canada goose and mute swan

ovum_diameter_mm = actual ovum diameter (mm) calculated from log values in Birkhead et al. 1994, or measured in the current study (canada goose and mute swan)

ovum_SA_mm2 = ovum surface area, extrapolated from diameter under the assumption that the ovum is spherical (4πr^2)

log_total_sperm = the log of the total number of sperm and holes (summed) reported by Birkhead et al. 1994 (read directly from plots) or measured in the current study (canada goose and mute swan)

log_holes = the log of the total number of holes only, reported by Birkhead et al. 1994 (read directly from plots) or measured in the current study (canada goose and mute swan)

total_sperm_ovum = actual total number of sperm and holes (summed) on the entire ovum, calculated from the log values reported in Birkhead et al. 1994

holes_ovum = actual number of holes only on the entire ovum, calculated from the log values reported in Birkhead et al. 1994