Why do eggs fail? The problem of unhatched eggs

Ashleigh Marshall

An in-depth exploration of the problems associated with hatching failure in birds, and the methods used to identify it, ahead of the Science and Conservation Event “Why do eggs fail?” on Tuesday 11 May.

Reproduction can be a costly process, representing a massive amount of investment on behalf of the parents, but producing offspring which go on to reproduce themselves is also often the only source of evolutionary fitness for many organisms. It follows that reproductive failure should be strongly selected against, and hence should be reasonably uncommon. However, in birds, on average 10% of all eggs laid fail to hatch, with much higher rates occurring in some populations of threatened species. For example, the California Condor (Gymnogyps californianus), Little Spotted Kiwi (Apteryx owenii), and Pink Pigeon (Nesoenas mayeri) have all experienced hatching failure rates of around 65%.

Image 1: Little Spotted Kiwi foraging at Zealandia EcoSanctuary, Wellington, New Zealand © Judi Lapsley Miller, CC BY 4.0 , via Wikimedia Commons
Image 1: Little Spotted Kiwi foraging at Zealandia EcoSanctuary, Wellington, New Zealand © Judi Lapsley Miller, CC BY 4.0 , via Wikimedia Commons

While many eggs may be lost to external factors such as predation, extreme weather, or abandonment, all of which can be understood and often even managed to some extent, questions remain around eggs that are incubated to term successfully but still fail to hatch. Suggested causes of these failures include environmental, genetic, disease-related, and behavioural drivers. Failed eggs have either experienced embryo mortality (where the embryo dies at some point between fertilisation and hatching), or they were never fertilised in the first place. However, eggs in this latter group can sometimes be hard to identify. If an embryo dies before visible signs of development become apparent, usually around 72 hours after fertilisation, the egg may be almost indistinguishable visually from an unfertilised egg and may be misidentified as having experienced fertilisation failure. Similarly, eggs which have failed to hatch can start to break down and become ‘addled’, which can disguise the earliest signs of development and cause such eggs to also be mislabelled as ‘infertile’. As different drivers, or combinations of drivers, are likely to result in an egg failing to be fertilised versus experiencing embryo mortality, it is important to establish the fate of an unhatched egg, in order to identify the potential drivers and apply the most appropriate mitigation strategies.

Image 2a (Left): Addled egg © Nicola Hemmings; Image 2b (Right): A canary embryo from an egg that failed during incubation, viewed through a microscope © Ashleigh Marshall
Image 2a (Left): Addled egg © Nicola Hemmings; Image 2b (Right): A canary embryo from an egg that failed during incubation, viewed through a microscope © Ashleigh Marshall

In a method using fluorescence microscopy, the germinal disc (a white spot on the surface of the yolk where the embryo will develop from if the egg is fertilised) and perivitelline layer (a transparent layer surrounding the yolk) are stained with Hoechst dye and examined under a microscope (Image 3). This method enables an investigator to locate sperm cells (to determine if sperm reached and penetrated the ovum – Image 4) and embryonic cells (to determine if fertilisation and embryogenesis – the development of the embryo – occurred). One study looking at eggs diagnosed as ‘infertile’ from five endangered species used such fluorescence microscopy to show that only 26% of these eggs had actually experienced fertilisation failure. Another study applying these methods to eggs of Blue Tits and Great Tits showed that with non-microscopic inspection alone, 50% and 32% of unhatched eggs respectively could have been misidentified as being unfertilised.

Image 3a (Left): Microscope set-up for dissecting an egg; Image 3b (Top Right): An egg yolk with a visible germinal disc (white circle); Image 3c (Bottom Right): Embryonic cells on the germinal disc viewed under a fluorescence microscope © Nicola Hemmings
Image 3a (Left): Microscope set-up for dissecting an egg; Image 3b (Top Right): An egg yolk with a visible germinal disc (white circle); Image 3c (Bottom Right): Embryonic cells on the germinal disc viewed under a fluorescence microscope © Nicola Hemmings

Image 4a (Left): Sperm on the perivitelline layer viewed under a fluorescence microscope © Nicola Hemmings; Image 4b (Right): Holes in the perivitelline layer made by sperm penetrating the ovum © Nicola Hemmings
Image 4a (Left): Sperm on the perivitelline layer viewed under a fluorescence microscope © Nicola Hemmings; Image 4b (Right): Holes in the perivitelline layer made by sperm penetrating the ovum © Nicola Hemmings

Very few studies accurately distinguish between fertilisation failure and embryo mortality using these, or similar, methods, but their results show that the assumption that undeveloped eggs are unfertilised is unreliable. This could have implications for our overall understanding of life history in wild populations, but has perhaps even more serious implications for both captive and free-living managed populations. For example, captive-breeding programmes of threatened birds often carefully monitor the breeding success of pairs and apply management interventions to mitigate issues and improve reproductive outcomes. A pair consistently laying ‘infertile’ eggs may end up being split up and paired with new mates based on the assumption that the male is not producing sperm or is otherwise having issues successfully fertilising the eggs. However, if these eggs are not actually unfertilised, but have instead experienced embryo mortality, a different issue may be at play which might not be resolved by remating the birds. It is similarly important to accurately establish a male’s fertility status through the presence or absence of sperm in eggs during selection of birds for a translocation event, as inclusion of an infertile male could threaten the translocation’s success.

Image 5: Male hihi © Martin Sanders
Image 5: Male Hihi © Martin Sanders

Recent applications of these fluorescence microscopy methods include examining unhatched eggs of the Critically Endangered Kākāpō (Strigops habroptilus) in which 61% of eggs fail to hatch. 72% of visually undeveloped eggs were found to be fertilised, leading to reassessment of the previous attribution of undeveloped eggs to male infertility. The methods are also being applied to the Vulnerable Hihi (Notiomystis cincta), which has undergone a series of translocations to secure predator-free islands resulting in the successful establishment of multiple populations. A recent management question for this species regards whether gene flow can be ‘topped up’ or maintained between these separate populations by moving eggs, rather than adults or juveniles. As Hihi experience a hatching failure rate of 35% this work will help to capture the baseline information needed to model how plausible such egg translocations could be, as well as the impact on both donor and recipient populations.

Female Hihi © Vix Franks
Female Hihi © Vix Franks

Join the upcoming ZSL Science and Conservation Event ‘Why do eggs fail?’ on Tuesday 11 May to hear more on this topic from world experts, including some of the reasons that lead to female reproductive failure, and how the methods discussed might be beneficial to the captive-breeding programmes which play a key role in conservation of threatened species.

Click here for event information

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