Unhatched eggs: methods for discriminating between infertility and early embryo mortality

Abstract

Unhatched eggs are a common phenomenon in birds and are often referred to as being ‘infertile’, which (confusingly) can mean at least two things: (1) that the ovum has not been fertilized or (2) that the embryo has died during development. These two broad categories of hatching failure can be difficult to distinguish, particularly in the early stages of embryo development. We describe methods to distinguish between infertility (due to insufficient sperm) and early embryo mortality in passerine eggs using the Zebra Finch Taeniopygia guttata as a model. We also describe how we successfully adapted these methods for use on eggs from a wild species, the Tree Sparrow Passer montanus, collected after the incubation period, and show that sperm can be visualized on the perivitelline layer of unhatched eggs even several weeks after laying.

Low fertility of wild hybrid male flycatchers despite recent divergence

“…When possible (i.e. when present and not degraded, n ¼ 8), the perivitelline layer of unhatched eggs was dissected and scanned for sperm using fluorescence microscopy [11]. The data were deposited in the Dryad repository: http://dx.doi.org/10.5061/dryad.5d44q [12].…”

Inbreeding causes early death in a passerine bird

“…All eggs were fostered to unrelated parents on the day they were laid. We used the techniques described by Birkhead et al 21 to distinguish between fertile and infertile eggs. Infertile eggs are those where a sperm pronucleus has not fused with the female pronucleus, and therefore no zygote is formed.…”

Causes of hatching failure in endangered birds

“…For confirmation of fertilization, the germinal disc (GD) was stained with Hoechst 33 342, a fluorescent DNA marker, and examined under a fluorescence microscope for the presence of embryonic cells [11]. Eggs were scored as either (i) infertile (no cell nuclei in the GD) or (ii) fertile but having suffered embryo mortality (cell nuclei in the GD and sperm on the PVL).…”

“…All of the perivitelline layer (PVL) found in each sample was cleaned thoroughly and examined for the presence of sperm, following the procedure described in the study of Birkhead et al [11] (which was validated for incubated eggs; see the electronic supplementary material). Sufficient material was obtained from all eggs to determine their fertility status, and for fertile eggs where the entire PVL was retrieved (63 hihi, 16 helmeted honeyeater (four wild and 12 captive), 37 orange-bellied parrot and 17 Spix's macaw eggs) the total number of sperm was counted.…”

“…Simple macroscopic examination (as described above) will therefore over-estimate infertility. Here, we use new methods [11] to determine the fertility of, and number of sperm in, unhatched eggs from five endangered species.…”