An evaluation of different methods for assessing eggshell pigmentation and pigment concentration using great tit eggs

Abstract

The striking variation in colour and maculation of bird eggs has fascinated biologists since centuries, and many hypotheses based on mechanical, physiological or signalling functions have been proposed for its evolution by natural and sexual selection. Protoporphyrin is the main eggshell pigment found in brown maculated eggs, and is assumed to function as a mediator of these selection processes. It is a precursor of heme with pro‐oxidant properties, and hence a link between brown maculation and female condition has been proposed and tested in a number of studies, albeit with contrasting results. A variety of different visual methods have been used to quantify outer eggshell pigmentation, which has been assumed to correspond to overall quantity of protoporphyrin in the shell. Yet, this relationship has rarely been tested. The aim of this study was to apply four commonly used methods to assess pigmentation in great tit eggs with protoporphyrin as the predominant eggshell pigment, and to compare the results of these methods. Specifically, we 1) ranked eggshell pigmentation by human naked eye, 2) applied a granularity approach and 3) measured spectrophotometric reflectance of eggshell pigments. Second, we estimated the relationship between outer eggshell pigmentation (i.e. estimated by three different methods above) and true protoporphyrin concentration deposited in the entire shell measured by HPLC. Among‐method estimates were significantly correlated for the traits describing pigment ‘darkness’ only. While the model including scores based on human naked eyes explained 16% of the variance of pigment concentration in the entire shell, spectrometry explained 27%, and the granularity approach explained 40%. Thus, the estimation of true pigment concentration in the entire shell from the visible outer side of the shell is most reliable with the granularity approach. It is relevant for studies where the maintenance of the integrity of the eggs is essential.

Pigmentation of White, Brown, and Green Chicken Eggshells Analyzed by Reflectance, Transmittance, and Fluorescence Spectroscopy

“…Brown eggshells begin to absorb at λ <750 nm by irradiation through the outer surface side and produce a nice vibronically structured reflectance spectrum, as shown in Figure (a). The overall absorption strength varies somewhat with the flock and age of the hens but the vibronic structure does not change . Irradiation from the inner surface side exhibits the same vibronic structure in the Vis range, but with very low absorption strength, Figure (b).…”

“…The overall absorption strength varies somewhat with the flock and age of the hens but the vibronic structure does not change. [36] Irradiation from the inner surface side exhibits the same vibronic structure in the Vis range, but with very low absorption strength, Figure 4 (b). Hence, the Vis-pigment must be non-uniformly distributed over the shell depth.…”

“…[32] The representation of eggshell colors in the avian tetrachromatic color opponent space is practiced. [33,34] Optical spectroscopy is furthermore beneficial for the separation of specular from diffuse reflectance with emphasis on highly glossy specimens, [35] for the investigation of reflectance spectra under the aspect of correlation with the pigment concentration, [3,[36][37][38] as well as for the aspect of the pigment stability against strong photoirradiation. [39] Maurer et al compared the spectrally resolved transmitted light through wild bird eggshells to their reflectance values.…”

SpotEgg: an image‐processing tool for automatised analysis of colouration and spottiness

“…However, this method has low inter-observer repeatability Brulez et al (2014a) as it uses researcher' skills to visually quantify three scores (visual expert judgement): spot pigment intensity, distribution of spotting over the surface and average spot size. Furthermore, other studies suggest that these scores are not a good proxy indicator for pigment quantification (Brulez et al 2014b, Wegmann et al 2015 which may be result of its low repeatability.…”

Connecting the dots: exploring the relationship between avian eggshell pigmentation and paternal provisioning effort

“…Because visual scoring of egg pigmentation is not always reliable (Brulez et al, 2014a,b;Stoddard et al, 2012;Wegmann et al, 2015), we also took digital photographs of each egg before swapping. All photos were taken in the field on a standard black background from approximately 15cm away (lighting conditions varied, but efforts were made to take all photos in moderate shade).…”