Including the Invisible Fraction in Whole Population Studies: a Guide to the Genetic Sampling of Unhatched Bird Eggs

Abstract

1. Early embryo mortality has recently been proven to be a significant component of avian reproductive failure. Due to the difficulty in distinguishing eggs which have suffered early embryo mortality from unfertilised eggs, this cause of reproductive failure has historically been underestimated and overlooked. 2. We describe methods for recognising and collecting undeveloped, unhatched eggs from wild bird populations, identifying and isolating embryonic material in unhatched eggs, and efficiently extracting DNA from those samples. We test these methods on unhatched eggs collected from the field which have undergone post-mortem incubation. 3. We obtained DNA yields from early-stage embryos that are sufficient for a wide range of molecular techniques, including microsatellite genotyping for parentage analysis and sex-typing. The type of tissue sample taken from the egg affected downstream DNA yields and microsatellite amplification rates. Species-specific microsatellite markers had higher amplification success rates than cross-species markers. We make key recommendations for each stage of the sampling and extraction process and suggest potential protocol improvements and modifications. 4. Genetic and possibly genomic analysis of embryos that die early in development has the potential to advance many fields. The methods described here will allow a more in-depth exploration of the previously overlooked causes of early embryo mortality in wild populations of birds, including threatened species.

Genotyping blastoderms of avian eggs

“…We improve on this method by genotyping the fertile blastoderms of unincubated and slightly incubated eggs to provide additional genetic information such as parentage as well as retain the PVL for sperm observations (see also Morland et al, 2023).…”

“…Overall, 10% (68/707) of the genotyped eggs had such extra alleles (Table 3). Morland et al (2023) F I G U R E 1 Number of sperm counted on the PVLs of experimental and non-experimental eggs. To improve readability, we excluded one experimental egg that had an exceptionally high sperm count (2724).…”

“…A recent study on the endangered hihi, Notiomystis cincta (Morland et al, 2023) describes a method similar to ours for genotyping solely unhatched eggs.…”

“…Other methods described include sucking up the blastoderm with a pipette (Strausberger & Ashley, 2001), or simply cutting out the blastoderm and PVL (without filter paper; Egloff et al, 2009;Morland et al, 2023). These methods provide a faster, simpler technique for removing the blastoderm, but the main disadvantage is that the PVL over the GD-an area that tends to contain higher numbers of sperm relative to the rest of the PVL (Birkhead et al, 1994) does not remain intact.…”

“…These methods provide a faster, simpler technique for removing the blastoderm, but the main disadvantage is that the PVL over the GD-an area that tends to contain higher numbers of sperm relative to the rest of the PVL (Birkhead et al, 1994) does not remain intact. Another disadvantage is that including the PVL in the DNA sample can increase the risk of contamination of parental DNA, in particular when embryo DNA concentrations are very low Morland et al, 2023).…”