The Fourth Egg Explained

For today’s blog post I’m handing over to Dr. Nicola Hemmings, from the Department of Animal and Plant Sciences here at the University of Sheffield.  Nicola, to whom I give a really big vote of thanks for her work here, is an expert on the causes behind hatching failures and has had a look at the fourth egg removed (under licence) during the ringing…   Over to Nicola:

Those of you who followed the Sheffield Peregrines last year (2015) will remember that two out of four eggs didn’t hatch. These eggs were collected during chick ringing, and I was able to examine them in my lab at the Department of Animal & Plant Sciences, University of Sheffield, to determine the causes of hatching failure. To learn more about my findings last year, you can look at the archived post here

This year, things went a little better for the peregrine eggs – three out of four hatched, leaving only one egg left behind.

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When the chicks were ringed (Monday 16th May), the egg was collected so that we could once again try to identify why it failed to hatch.

I received the egg in rather amusing protective packaging –this was one egg you certainly wouldn’t want to bite into!

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As I opened the container, I could smell that familiar stench. Remember, this egg had been sat in a warm, humid nest for about seven weeks. It had undergone a full incubation period, followed by several weeks of being sat (and excreted) on by the chicks and their parents. It was covered in faeces, feathers, and other debris. There was no doubt its contents would be severely degraded.

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Despite this, I wasn’t quite ready for extreme level of deterioration I encountered on opening the egg – even I have rarely seen such putrid egg contents! As the rich green soup poured out, my eyes began to water. My research colleagues were repulsed by the foul fumes that filled the lab.

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I must admit I was concerned this egg was beyond examination. However, with characteristic determination, I began steadily to sift my way through the gunk, pouring subsample after subsample of the soup into a new dish and diluting it with saline solution to help clear the fluid.

I wasn’t simply filled with blind hope; I had good reason to think I might find something interesting or useful in among this smelly concoction. I’ve examined many unhatched eggs – probably several thousand – over the years, as part of my research into the causes of hatching failure in birds, so I am used to finding golden bits of information among a rotten mess! I also conducted an experiment a few years ago (using turkey eggs) to see how long non-developing eggs could be incubated before I was no longer able to get useful information from them. Even after several weeks of intensive incubation, I was still able to find degraded embryonic tissue, as well as sperm cells embedded in the layer surrounding the egg yolk. It’s amazing how much you can learn from a rotten egg!

Unhatched egg examinations are an important tool for bird conservation. Across species, around 10% of all eggs fail to hatch, but in some critically endangered species the failure rate is over 70% – that’s almost three-quarters of all potential chicks never hatching! By looking at unhatched egg contents, we can diagnose fertility problems (i.e. insufficient sperm), detect egg infections, and identify abnormalities in developing embryos that may be linked to genetic problems or environmental factors. Understanding these causes of hatching failure allows rapid and effective measures to be put in place to improve success in the next breeding season.

Back to the peregrine egg. Despite the extreme degradation, my long-practiced egg examination skills didn’t fail me. In one diluted subsample, I suddenly noticed a tiny lump, surrounded by what looked like a thin membranous sack.

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On closer inspection, the “lump” was very obviously a tiny peregrine embryo that had begun developing, but then died at a relatively early stage. The photo below shows the embryo, separated from its “sack”, next to a 1 cm square grid for scale (each of the smallest squares on the grid = 1mm). The “sack” is in fact a glycoprotein layer that surrounds the yolk, called the perivitelline layer. When you dunk a bread soldier into your fried egg, it’s the perivitelline layer that ruptures to allow the yolk to ooze out. The perivitelline layer attaches the yolk to the embryo, which is important because the yolk is the embryo’s only source of nutrition throughout the entire developmental period. Remains of the other extra-embryonic membranes may also be present: the amnion, which surrounds the embryo and secretes amniotic fluid to prevent the embryo from drying out; the allantois, which provides a gas-exchange surface so the embryo can breathe, and also removes waste; and the chorion, which serves as a protective membrane for all the embryonic structures.

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It is possible to roughly estimate the “age” at which an embryo died (i.e. how long it had been incubated for before death) based on its developmental stage. The developmental stages of the domestic chicken embryo are well-established, having been described by Hamburger & Hamilton (1951) using morphological landmarks. You can see the full developmental series  here, and read the original paper if you are interested. These stages have also been described for a few other bird species – in general, early development proceeds at a very similar rate across species. I therefore used Hamburger & Hamilton’s staging series as a guide when examining the peregrine embryo.

If you look closely at the embryo, you can clearly see that the eye is pigmented and the limb buds are slightly elongated. This corresponds with stages 20-23 of Hamburger & Hamilton’s series i.e. approximately 3-4 days development. It is difficult to give a more precise estimate of the developmental stage, because some of the fine morphological structure of the embryo had degraded.

Embryo mortality in birds is most common very early (first three days) or very late (just before hatching) in incubation. This peregrine embryo died at the tail-end of the critical early development period. In these early stages, death mainly results from problems with the formation of organs and the respiratory system, which may result from interference during incubation (e.g. eggs being exposed to abnormal temperatures for prolonged periods). It would be interesting to look back at the nest camera footage (and weather conditions) 3-4 days after incubation started, to see if there are any clues about what went wrong [ed – the temperature dropped to 1.5ºC on the early morning of 28th March and the night of March 30th, with the clutch haivng been completed on March 26th ; the cold tempratures do coincide with this 3-4 day timeframe, but it’s not possible at present to look back to see if there was a period when an egg was left uncovered for any length of time].

It is worth noting that prior to the onset of incubation eggs remain in a state of suspended animation. The embryo undergoes some development (rapid cell division) inside the mother’s body before the egg is laid, but assuming ambient temperatures are below ~24ºC, development pauses after egg-laying until incubation begins. In birds, 24ºC is considered to be to “physiological zero” – below this threshold no development occurs. For UK breeding birds, this makes timing of incubation and chick hatching fairly straightforward. For example, blue tits (Cyanistes caeruleus) can lay up to 16 eggs (usually 8-12) at a rate of one egg per day. However, the female only begins incubating after the ultimate or penultimate egg is laid (as do the Peregrines), ensuring that all the chicks hatch over the course of just one or two days. The pre-incubation period poses a bigger problem for birds in hotter climates, where ambient temperatures frequently exceed physiological zero. If the egg temperatures rise above 24ºC, but do not reach full incubation temperature (36-38ºC), developmental abnormalities will occur and the embryo is likely to die. As a result, birds that breed in hot environments often have smaller clutches and begin incubating as soon as the first egg is laid, leading to asynchronous chick hatching.

I have now stored the embryo, along with some other samples from the egg, in 100% ethanol for DNA analysis. The molecular lab here in Sheffield are hoping to find funds to pay for this work, which may reveal whether or not we have a new peregrine female this year.

For now, I will leave you with a stark reminder about why you should always store your eggs somewhere cool and dry, and avoid leaving them too long before eating them. This is what was left of the once-nutritious yolk, after seven weeks of being sat on by a peregrine falcon. Delicious.

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  1. A fantastic report which greatly appealed to the scientific side of my brain but which made me sad for the loss at the same time!

  2. A really interesting read. Thanks a lot for sharing.
    Hope the funds for the DNA analysis can be found, as it would be great to find out if we have a different female this year.

  3. Totally fascinating, but I am happy not to have been present for the unshelling!
    The photos are brilliant and the explanations of what everything is are very clear.
    Thank you so much

  4. Fantastic reading, and very well described. I had been waiting to find out why the 4 th egg didn’t hatch.

    Totally loving this, thank you Sheffield peregrines.

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