Eggshell spot scoring methods cannot be used as a reliable proxy to determine pigment quantity

Abstract

Eggshell maculation of most passerines is due to the deposition of the pigment protoporphyrin which is produced during biosynthesis of blood haem. Its functional signifi cance has only received empirical attention in recent years. Th is interest has generated a number of hypotheses of which some remain untested partly because the quantifi cation of protoporphyrin is analytically challenging and can be prohibitively expensive. Many studies have therefore used the extent of eggshell spotting as a proxy for total eggshell protoporphyrin concentration, although this has not been formally tested. Pigment scoring involves recording visible eggshell pigment attributes, such as spot intensity, distribution and size. Since even immaculate eggs can contain some protoporphyrin, there remains doubt over the degree to which visible pigment correlates with total pigment content of the shell. In this study, we test whether visible pigment scoring can be used as a proxy for protoporphyrin concentration of an eggshell. We use pigmented eggshells of two common British passerine species to compare eggshell spot intensity, distribution and spot size (as used by the visual pigment scoring method) with direct measures of eggshell protoporphyrin concentration. In addition, we compared an alternative method of pigment scoring, the pixel pigment scoring method, using a computer programme to quantify the number of pixels exceeding a specifi ed colour threshold. We demonstrate that although results from both scoring methods were positively correlated with eggshell protoporphyrin concentrations, the correlations were not suffi ciently strong to be used as surrogates in studies where actual pigment concentrations are required.

Darker eggshell spotting indicates lower yolk antioxidant level and poorer female quality in the Eurasian Great Tit (Parus major)

“…Similar to our results, Holveck et al (2012) found that the spectral chroma of brown spots was significantly correlated with spot intensity and spot size, but not with spot distribution, in a French population of Great Tits. Brulez et al (2014) reported that eggshell protoporphyrin concentration was correlated with spot intensity and spot size, but not with spot distribution, in British Great Tits. However, the authors pointed out that correlations with pigment concentration were not sufficiently strong to be used as reliable surrogates, and in our study we found similarly moderate correlation coefficient values.…”

“…Others determined pigment concentration of the eggshell by chemical analysis (Maurer et al 2011b, Cassey et al 2012, Duval et al 2013, Brulez et al 2014, or analyzed digitized pictures of eggshells to determine the percentage of spotted eggshell surface (Martínez-de la Puente et al 2007, Sanz and García-Navas 2009, Bulla et al 2012, Holveck et al 2012, Brulez et al 2014), or to determine hue, saturation, and brightness derived from HSB and RGB (Martínez-Padilla et al 2010, Cassey et al 2012, Holveck et al 2012), or to determine other spotting pattern variables, such as pattern contrast and marking size (Stoddard et al 2012). Finally, many studies used visual scoring of photographs, and spotting pattern was expressed as spot intensity, spot size, and spot distribution (Gosler et al 2005, Sanz and García-Navas 2009, López de Hierro and De Neve 2010, Bulla et al 2012, Holveck et al 2012, De Coster et al 2013, Hargitai et al 2013, Brulez et al 2014.…”

Eggshell spotting does not predict male incubation but marks thinner areas of a shorebird's shells

“…This is a novel and important finding with regard to the developmental biology of the quail (and presumably other Galliformes), suggesting that protoporphyrin may also be accumulated by way of an increase in eggshell thickness. Presumably, therefore, the absolute pigment content cannot be evaluated correctly by any attempt at a visual description of an eggshell's external pigmentation (e.g., Brulez et al 2014, Pike 2015. It thus appears that this overlooked mechanism of pigment deposition through a thickening of its layer can better explain some conflicting results published earlier-for example, that the greater number of visible brown spots on quail eggshells is not due to enhanced protoporphyrin deposition (Duval et al 2016).…”

A Machine Vision System for Detecting Fertile Eggs in the Incubation Industry

“…Otsu algorithm has been widely used in previous related work such as approaches in Refs. 10,12,13,17 . This algorithm exhaustively searches for the threshold that minimizes the intra-class variance defined as a weighted sum of variances of the two classes.…”

“…Some of the research is dedicated to the detection of cracks and dirt on eggs, 4,5,6,7,8,9,10,11,12,13 or measuring physical coordinates of eggs 5,11,14,15,16,17 . In a similar manner to the candling machines, most of these researches usually use light sources such as the incandescent lamp, fluorescent lamp or halogen lamp to back-illuminate the egg.…”