DOI: 10.5061/dryad.vmcvdncqr
title: Numbers of sperm associated with the perivitelline layer of seabird (and other bird species) eggs

description: 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.
description: We obtained eggs of six single-egg clutch seabird species, from
 two unrelated taxa, the Alcids (Common Guillemot <i>Uria
 aalge</i> (N = 5), Puffin <i>Fratercula arctica</i> (N =
 2), and Razorbill <i>Alca torda</i> (N = 5)), and the
 Procellariiformes (Manx Shearwater <i>Puffinus puffinus</i> (N
 = 3), Northern Fulmar <i>Fulmarus glacialis</i> (N = 2), and
 Storm Petrel <i>Hydrobates pelagicus</i> (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 cm<sup>2</sup> 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.
description: <b>Below are descriptions of each data column in this
 dataset, separated by sheet</b> Sheet 1:
 "<b>seabird sperm data (this study</b>)"
 <b>common_name</b> = species common name
 <b>species</b> = species binomial name
 <b>spp_no_this_study</b> = the number allocated to
 each species in the current study (e.g. in plots)
 <b>ovum_diameter_mm</b> = ovum diameter in mm (note
 this is a mean value for each species because not all ova could be
 measured) <b>ovum_SA_mm2</b> = ovum surface
 area, extrapolated from diameter under the assumption that the ovum is
 spherical (4πr^2) <b>GD_stage</b> =
 developmental stage of the germinal disc, according to the normal stages
 of chick development (Hamburger & Hamilton 1951)
 <b>est_development_hrs</b> = estimated number of
 hours of development the embryo had undergone before egg collection, based
 on the germinal disc developmental stage
 <b>gd_sperm_50mm2</b> = the number of sperm counted
 in a 50mm^2 section of perivitelline layer from the germinal disc pole of
 the ovum <b>gd_holes_50mm2</b> = the number
 of holes counted in a 50mm^2 section of perivitelline layer from the
 germinal disc pole of the ovum
 <b>vp_sperm_50mm2</b> = the number of sperm counted
 in a 50mm^2 section of perivitelline layer from the vegetal pole of the
 ovum <b>vp_holes_50mm2</b> = the number of
 holes counted in a 50mm^2 section of perivitelline layer from the vegetal
 pole of the ovum
 <b>mean_total_sperm_50mm2</b> = the mean number of
 sperm and holes (summed) counted per 50mm^2 section of perivitelline layer
 (mean of germinal disc and vegetal pole counts)
 <b>total_sperm_ovum</b> = 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
 <b>mean_holes_50mm2</b> = the mean number of holes
 only, counted per 50mm^2 section of perivitelline layer (mean of germinal
 disc and vegetal pole counts)
 <b>holes_ovum</b> = 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: "<b>Birkhead et al 1994
 data</b>" <b>common_name</b> =
 species common name species = species binomial
 name <b>spp_no_this_study</b> = the number
 allocated to each species in the current study (e.g. in plots)
 <b>species_no_Birkhead1994</b> = 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)
 <b>log_ovum_diameter_mm</b> = 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
 <b>ovum_diameter_mm</b> = actual ovum diameter (mm)
 calculated from log values in Birkhead et al. 1994, or measured in the
 current study (canada goose and mute swan)
 <b>ovum_SA_mm2</b> = ovum surface area, extrapolated
 from diameter under the assumption that the ovum is spherical
 (4πr^2) <b>log_total_sperm</b> = 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) <b>log_holes</b> = 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) <b>total_sperm_ovum</b> =
 actual total number of sperm and holes (summed) on the entire ovum,
 calculated from the log values reported in Birkhead et al. 1994
 <b>holes_ovum</b> = actual number of holes only on
 the entire ovum, calculated from the log values reported in Birkhead et
 al. 1994