Since we removed the unhatched eggs from the nest when the chicks were ringed, I’ve been curious to know what we might be able to learn as to the reasons behind their failure. We’ve been very fortunate to be able to enlist the help of Dr Nicola Hemmings of the Department of Animal and Plant Sciences here at the University of Sheffield, and she has now had time to analyse the eggs. Below is a report she’s written especially for the blog that expands on the brief notes posted to Twitter: great work Nicola!
Causes of hatching failure in Sheffield’s urban Peregrines
Dr Nicola Hemmings
On 03 June 2015, I examined two failed eggs from the 2015 Sheffield Peregrines clutch in an attempt to understand why the eggs had not hatched. The eggs were collected for analysis under license on 15 May, when Sorby Breck Ringing Group went to ring the chicks that had hatched from the other two eggs. They were then stored in a refrigerator for just over two weeks before analysis at the Department of Animal & Plant Sciences, University of Sheffield.
Hatching failure is a common problem for birds and something that I have studied for a number of years. In general, a small proportion of eggs (i.e. 1-2%) remain unhatched, but occasionally entire clutches fail. The extent of hatching failure varies markedly across birds and tends to be higher in endangered species. This year, the Sheffield Peregrines experienced a fairly high level of loss: 50% hatching failure.
Hatching failure results from two main causes: (1) the egg fails to be fertilised (infertility) or (2) the fertilised egg fails to hatch (embryo death). These two causes of hatching failure are driven by different underlying factors, so it is useful to distinguish between them. In addition, the developmental stage at which embryo death occurs can provide insight into what went wrong. Distinguishing between these different causes of hatching failure requires specialised techniques: I’ve provided some links to additional reading (see at the end of this report) in case you would like to learn more.
The first Peregrine egg felt light, indicating that it was degraded and may have even dried out slightly. This often happens if there is a slight crack or hole in the shell. The egg opened with a ‘pop’ and the unpleasant aroma of rotten egg filled the air! Inside the egg’s soupy contents was an obvious but badly deteriorated embryo. It was incredibly difficult to determine an exact developmental stage because most of the embryo’s distinctive features had turned to mush. However, the embryo was approximately 2-3 cm long and one fairly large limb was recognisable, suggesting that the embryo must have been developing for at least a week before it died. There were no late-stage features such as feathers and claws, so it is unlikely the embryo was older than approximately two weeks.
The second egg required more careful examination. On opening, it wasn’t as ‘soupy’ as the first egg and didn’t smell quite as bad. There was no obvious sign of development but the yolk had collapsed so it was impossible to locate the germinal disc (where embryo development begins). Looking for further clues, I fished out from the mixture a crucial component of the egg – the perivitelline layer (PVL). The PVL is a thin layer surrounding the egg yolk, almost like a clingfilm bag. It is made up of glycoproteins and actually consists of two layers stuck together: the ‘inner’ and ‘outer’ PVL. The inner PVL binds the yolk from the start, and as part of the process of fertilisation, sperm must dissolve a hole through the inner PVL, using enzymes stored in their head. Once inside, one sperm fuses with the female pronucleus – located in the germinal disc – after which development begins, fuelled by the nutritious yolk. Just minutes after fertilisation has occurred, the outer layer of the PVL forms around the yolk, trapping all the other sperm surrounding the egg at the time. The sperm are firmly stuck in this layer and the whole thing is very resilient to degradation. Therefore, if I take the PVL from an unhatched egg and look at it under the microscope, I can see if any sperm managed to get to the egg. This allows me to decide whether or not the egg was fertilised.
(Microscope image of sperm from the Peregrine egg. The fluorescent DNA dye is visible in the sperm’s head, which measures about 5 microns, or 0.005 mm. The tail is visible to the ‘south-west’)
I found numerous sperm on the PVL of the second unhatched Peregrine egg– a total of 42 sperm were found in a single 1cm2 sample. There were also lots of large clumps of ‘normal’ cells on the PVL, which were probably traces of embryonic tissue. There was no sign of an observable embryo, nor any trace of blood vessels, so in contrast to the first embryo, this embryo died very early on, probably in the first day or two of incubation when it was little more than a tiny disc of rapidly dividing cells.
So why did these two Peregrine embryos die? It is very difficult to identify the exact cause of embryo death in wild birds such as the Sheffield Peregrines, since so many different factors can influence embryo development and survival. However, the fact that they died at such different developmental stages suggests that there was a different cause for each embryo. Early-stage embryos tend to be fairly resilient to changes in environmental conditions, such as cold temperatures or delayed incubation, so it is more likely that the early stage embryo had an inherent developmental problem. In the early stages of development, cells are dividing rapidly and rates of gene expression are at their highest. This means that there is much more scope for genetic mutations to arise, which change the DNA sequences that make up genes and therefore alter their expression. If this happens to genes that are essential for development, normal cell division and differentiation will be disrupted and the embryo will die. I cannot be certain this is what happened to the early-stage Peregrine embryo, but it is a common cause of early embryo death in birds.
The other embryo, which developed for at least one week, is much more likely to have fallen foul of the prevailing environmental conditions. As embryos grow larger and their organs begin to form, they become much more dependent on stable incubation conditions, including consistent temperature and humidity. If the egg was left exposed for too long, or was allowed to become too damp and dirty, the embryo may have died due to chilling or infection.
Finally, any damage – however minor – to either egg would have disrupted gas exchange and accelerated water loss and infection. These would almost certainly lead to embryo death, at any stage. Therefore it is possible that both embryos may have simply been victims of a small crack or hole in the eggshell.
Happily, there is no evidence that mum or dad have any significant fertility problems: plenty of sperm reached all eggs and they had a fertilization rate of 100%. Let’s hope that next year, conditions during the egg laying and incubation periods are more favourable, and they enjoy 100% hatching success too.
Birkhead, T.R., Hall, J., Schutt, E., & Hemmings, N. (2008). Unhatched eggs; methods for discriminating between infertility and early embryo mortality. Ibis 150: 508-517. Available here or via <http://onlinelibrary.wiley.com/doi/10.1111/j.1474-919X.2008.00813.x/full>
Hemmings, N., West, M. & Birkhead, T. R. 2012. Causes of hatching failure in endangered birds. Biology Letters doi: 10.1098/rsbl.2012.0655. Available here or via <http://rsbl.royalsocietypublishing.org/content/roybiolett/8/6/964.full.pdf>