Quantifying the functional disparity in pigment spot-background egg colour ICP-OES-based eggshell ionome at two extremes of avian embryonic development

Abstract

It is known that a developing avian embryo resorbs micronutrients (calcium and other chemical elements) from the inner layer of the eggshell, inducing thinning and overall changes in the shell's chemical composition. However, an aspect yet to be explored relates to the local changes in the multi-elemental composition (ionome) of the pigment spot and adjacent background colour regions of eggshells resulting from avian embryogenesis (with respect to two extremes of embryonic growth: the maternal level at the moment of egg laying, and after the completion of embryonic growth). To address this problem, we used inductively-coupled plasma optical emission spectrometry (ICP-OES) to establish the elemental profiles of microsamples from the cryptic eggs of Capercaillie Tetrao urogallus and Black Grouse Tetrao tetrix, representing the background colour and pigment spot regions of the shell. We then related these to the developmental stage of the eggs (non-embryonated eggs vs. post-hatched eggshells) and their origin (wild vs. captive hens). Our results show an apparent local disparity between the pigment spot and background colour regions in the distribution of chemical elements: most elements tended to be at higher levels in the speckled regions of the shell, these differences becoming less pronounced in post-hatched eggshells. The trends of changes following embryonic eggshell etching between the pigment spot and background colour shell regions were conflicting and varied between the two species. We hypothesized that one potential working explanation for these interspecific differences could be based on the variable composition of elements (mostly of Ca and Mg), which are the result of the varying thickness of the individual shell layers, especially as the relative difference in shell thickness in the pigment spots and background colour regions was less in Black Grouse eggs. Overall, this investigation strongly suggests that egg maculation plays a functional role in the physiological deactivation of trace elements by incorporating them into the less calcified external shell layer but without participating in micronutrient resorption. Our major critical conclusion is that all research involving the chemical analysis of eggshells requires standardized eggshell sampling procedures in order to unify their colouration and embryonic status.

Figure 1

Overall eggshell thickness (average ± 95% CI) measured over the entire longitudinal section of the egg (from the sharp pole to the blunt one): both non-embryonated and post-hatched shells of the cryptic eggs of Black Grouse Tetrao tetrix and Capercaillie Tetrao urogallus (depicted as miniatures) are always significantly thinner in the background colour shell region than in the adjacent pigmented spot. Note: The shells of all the Black Grouse and Capercaillie eggs were respectively − 2.67% and − 5.34% thinner in the background colour than the pigment spot region. In each case, the t-test for paired comparisons showed highly significant differences with the exact P value equal to 0; see Table S1 for the statistical treatment.

Figure 2

Ratios of elemental concentrations (based on pooled data for all the eggs—see Table S2 and Table S3) between the pigment spot and background colour regions of eggshells at two extremes of avian embryonic development, defined as non-embryonated eggs (maternal level) and post-hatched (resorbed) eggshells in cryptic eggs of Black Grouse Tetrao tetrix and Capercaillie Tetrao urogallus. The variation in elemental concentrations in the spot-background eggshell regions within the same eggs (for corresponding pairs of measurements) is illustrated in Fig. S1. Note: The red line (ratio = 1) signifies no difference in eggshell elemental concentration between the pigment spot and background colour region; the bars with ratios < 1 indicate higher elemental concentrations in the background colour region.

Figure 3

%Change (= Concentration_post-hatched − Concentration_{non-embryonated} × 100/Concentration_{non-embryonated}) in elemental concentrations in the background colour and pigment spot regions of eggshells following embryonic eggshell etching, defined as non-embryonated and post-hatched eggshells of Black Grouse Tetrao tetrix and Capercaillie Tetrao urogallus. For the elemental concentrations and sample sizes, see Tables S2and S3. Note: The bars with positive values indicate elevated elemental concentrations following embryonic eggshell etching, whereas those with negative values indicate decreases within a given shell region. Note that because of some differences in eggshell elemental concentrations related to the origin of the eggs (see “Results”), the trend in %Change calculated within the sample of eggshells from wild and captive Black Grouse and Capercaillie retains the same direction, although the magnitude (bars) of %Change becomes smaller (after recalculation of the data from Tables S4 and S5).