Author Topic: A brief review of scientific studies on eggshell color from the past decade  (Read 1639 times)

Lindsay Helton

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White eggshells:

A study identified that it is notable that the protoporphyrin content which creates brown eggshell color in the uterine tissue of white-egg laying birds is not dissimilar to that of brown-egg laying birds. White-shelled eggs are not necessarily devoid of pigment. (Sparks, 2011).

A study identified that the presence of biliverdin and/or protoporphyrin IX does not inevitably mean that an egg is colored. Both pigments were identified as being widespread. When found in eggshells, they can cause brown or blue–green coloration, but many white avian eggs contain the pigments in low levels. An extensive survey determined that pigment-free white eggs are rare. An egg containing these pigments could thus either be colored or not, depending on the concentration of pigments. (Wiemann et al., 2019).

Brown eggshells:

A study by Samiullah et al found that the major pigment in eggshells of brown-egg laying hens is protoporphyrin IX, but traces of biliverdin and its zinc chelates were also present. The pigment appears to be created in the shell gland. This study reflected that the protoporphyrin IX synthetic pathway is identified, but precisely where and how it is synthesized in the shell gland of the brown-egg laying hen is unclear. The pigment was found to be deposited onto all shell layers including the shell membranes, but most of it was concentrated in the outermost layer of the calcareous shell and in the cuticle. The extent of pigment deposition was impacted by the housing system, hen age, hen strain, diet, stressors, and certain diseases such as infectious bronchitis. (Samiullah et al., 2015).

A study analyzed three different types of hens that generated white, pink or brown eggshells. Their data revealed three ways in which eggshell color was genetically influenced. First, high‐level expression of CPOX generated more protoporphyrinogen and a brown eggshell color. In contrast, high expression of FECH likely converted more protoporphyrinogen into heme, reduced protoporphyrinogen levels within the eggshell and generated a light color. Second, heme transporters also affected eggshell color. High‐level expression of BCRP, HRG1 and FLVCR were associated with brown, white and generally lighter eggshell colors, respectively. Finally, protoporphyrin precipitation also affected eggshell color, high expression of both SLCO1A2 and SLCO1C1 were associated with brown eggshell color. They thus identified seven genes in which expression levels in different tissues were associated with eggshell color. (Jianfei et al., 2020).

A study found a negative association between plasma and feces protoporphyrin concentrations during egg laying and eggshell brown chroma. This indicates that an increased creation of protoporphyrin in the liver resulting in elevated plasma and feces protoporphyrin concentrations could inhibit eggshell protoporphyrin pigmentation by way of affecting enzymatic activities. They identified that circulating pigment levels may influence shell gland pigment synthesis, therefore linking the physiological level of the female to eggshell coloration. (Hargitai et al., 2017).

Blue eggshells:

A study reflected that green and blue‐green shells contain biliverdin, an oxidative ring opening product of PPIX with broad absorption in the blue and orange‐red region. The study found that small differences in the absorption width or maximum induce the change of the color impression from more green to more blue. The green shells typically contain PPIX in addition to BV, resulting in a dark color. The study identified that the color pigment protoporphyrin IX (PPIX) is embedded in the protein phase of all four shell types as highly fluorescent monomers, in the white and light green shells additionally as non‐fluorescent dimers, and in the brown and dark green shells mainly as non‐fluorescent poly‐aggregates. Green shell colors were identified as being formed from an approximately equimolar mixture of PPIX and biliverdin. (Ostertag et al., 2019).

A study in 2021 narrowed down SNPs to a region of ~ 653.819 Kb on GGA21 that harbors five candidate genes associated with eggshell color: AJAP1, TNFRSF9, C1ORF174, CAMTA1, and CEP104. The shell gland of chickens laying dark and light blue eggshell were examined for detection of mRNA expression of the five candidate genes. The results showed differential expression levels of these genes in the two groups. The specific function of these genes has not yet been defined clearly in chickens and further in-depth studies are needed to explore the new functional role in chicken eggshell blueness. (Ming-Yuan et al., 2021).

A study found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which reflects independent integration events in the blue-shelled chickens from the two continents. (Wang et. al, 2013).

A genome wide association study (GWAS) was conducted in Chinese Dongxiang blue-shelled chicken underlying four traits of blue eggshell pigments: quantity of biliverdin (QB), quantity of protoporphyrin (QP), quantity of total pigment (QT), and color density trait (CD). (Darwish et al., 2019).

Miscellaneous:

A study indicated that phylogenetic dependence must be considered in further explanations of the functional significance of avian eggshell color. (Wragg et. al., 2013).

A study identified that organic anion transporting polypeptide family members (including solute carrier organic anion transporter family, SLCO1C1, SLCO1A2, SLCO1B3 and LOC418189) may affect pigment transport within eggshells. (Chuanwei et al., 2014).

A study reflected that a female’s ability to handle oxidative stress influences pigment deposition in the eggshell. They found a positive relationship between eggshell pigment distribution and maternal and yolk antioxidant protection, indicating that eggshell pigmentation is a signal of female (and offspring) quality. (Giordano et al., 2015).

The effect of parasites on eggshell color:

A study found a correlation between spot darkness and laying order that weakened under high parasite load. Their study suggests that anemia in females due to parasites led to diminished protoporphyrin from disintegrated red blood cells and hence a decreased deposition of protoporphyrin. (De Coster et al., 2012).

Eggshell color as affected by carotenoids:

A study completed found that biliverdin, the pigment responsible for the blue‐green color of a eggshell, is a strong antioxidant and that only females with high antioxidant capacity can deposit higher concentrations of biliverdin as eggshell pigment. Antioxidants such as carotenoids and vitamins are also abundant in the egg yolk, which serve as nutrient reserves for the developing embryo. Eggshell color was positively related to the concentration and amount of carotenoids and vitamin E in the yolk. (Hargitai et al., 2013). Another study also found similar results and reflected that eggshell color intensity was positively related to the concentration and amount of carotenoids and vitamin E in the yolk. (Navarro, 2011).

A study found that throughout the laying sequence, biliverdin concentration increased while eggshell thickness, yolk carotenoid concentration, and lysozyme concentration in the albumen all decreased. The biliverdin concentration explained, at most 46% of the variation for all eggshell coloration metrics and suggest biliverdin concentration is a better predictor of egg quality than egg coloration in the birds studied. (Butler et al., 2015).

Morales et al completed a study in 2011 and found that there was a decline in eggshell color with laying order, indicating pigment limitation for females. However, carotenoid-supplemented females had increased second egg coloration compared to controls. Their results suggest that biliverdin-based eggshell coloration is costly to create and can be eased by carotenoid accessibility. (Morales et al., 2011).

Eggshell color as affected by temporal factors:

Eggshell color was studied on over 400 eggs at the moment of oviposition, at the onset of incubation, and at the end of incubation. Results showed that egg color faded significantly during the study period. This study indicates that egg coloration studies should account for temporal changes in egg coloration. (Moreno et. al, 2011).

A study identified that blue‐green chroma and brightness of the same eggs varied significantly between years. This implies the need for future research into the amount of chemical and physical deterioration of eggshell appearance even during relatively short‐term storage. (Cassey et al., 2011).

A study found high correlations between the shell color at 42 wk of age and subsequent ages (50, 60, and 70 wk) of laying hens, indicating that the intensity of eggshell color is more stable after egg-laying peaks. They also found that the intensity of brown eggshell color fluctuates greatly among the whole laying cycle. (Huijuan et al., 2018).

Eggshell color as affected by environmental conditions:

A study showed that eggshell pigmentation is not only affected by female body condition but is also shaped by the female’s early life experience such as stress exposure. Eggshell color was influenced by breeding conditions, which stresses the complexity of the relationship between eggshell pigment concentrations and environmental conditions. (Duval, 2014).

A study found that females who experience a decline in antioxidant capacity due to food limitation experience a reduction in blue‐green eggshell color. Food-restricted females laid eggs with significantly higher eggshell brown chroma, spot intensity, and protoporphyrin concentration. The results suggest that limitation in nutrient availability increases deposition of protoporphyrin into the eggshell. (Hargitai et. al, 2017).
Another study found that eggs with lower yolk antioxidant concentration had higher average eggshell brown chroma. Results from the study indicated that eggshell color is affected by female antioxidant and nutrient accessibility. (Hargitai et al., 2016).

A study identified that the blue‐green color of eggshells can disclose an inherent aspect of females' physiological condition, with only high‐quality females having adequate antioxidant capacity to pigment their eggs with large amounts of biliverdin. Subsequent work has argued instead that eggshell color might signal condition‐dependent traits based on diet. The study reflected that individual females contrasted strongly and consistently from one another, despite having been reared under uniform circumstances. (Dearborn et al., 2012).

Butler, M. Waite, H. Eggshell biliverdin concentration does not sufficiently predict eggshell coloration. Journal of Avian Biology Volume 47, Issue 4 p. 491-499A. First published: 19 November 2015 https://doi.org/10.1111/jav.00842

Cassey, P. Mark E. Hauber, Golo Maurer, John G. Ewen Sources of variation in reflectance spectrophotometric data: a quantitative analysis using avian eggshell colors. First published: 20 September 2011 https://doi.org/10.1111/j.2041-210X.2011.00152.x

Darwish HYA, Dalirsefat SB, Dong X, Hua G, Chen J, et al. (2019) Genome-wide association study and a post replication analysis revealed a promising genomic region and candidate genes for chicken eggshell blueness. PLOS ONE 14(1): e0209181. https://doi.org/10.1371/journal.pone.0209181

De Coster, G., De Neve, L. & Lens, L. Intraclutch variation in avian eggshell pigmentation: the anaemia hypothesis. Oecologia 170, 297–304 (2012). https://doi.org/10.1007/s00442-012-2304-1

Dearborn, D. Daniel Hanley, Katherine Ballantine, John Cullum, DeeAnn M. Reeder. Eggshell color is more strongly affected by maternal identity than by dietary antioxidants in a captive poultry system. Functional Ecology Volume 26, Issue 4 p. 912-920 Animal physiological ecology. First published: 18 May 2012 https://doi.org/10.1111/j.1365-2435.2012.02001.x

Giordano, M., Costantini, D., Pick, J.L. et al. Female oxidative status, egg antioxidant protection and eggshell pigmentation: a supplemental feeding experiment in great tits. Behav Ecol Sociobiol 69, 777–785 (2015). https://doi.org/10.1007/s00265-015-1893-1

Hamchand, R., Hanley, D., Prum, R.O. et al. Expanding the eggshell color gamut: uroerythrin and bilirubin from tinamou (Tinamidae) eggshells. Sci Rep 10, 11264 (2020). https://doi.org/10.1038/s41598-020-68070-7

Hargitai, R. Gergely Nagy, Márton Herényi, János Török. Effects of experimental calcium availability, egg parameters and laying order on Great Tit Parus major eggshell pigmentation patterns Ibis Volume 155, Issue 3 p. 561-570. First published: 18 June 2013 https://doi.org/10.1111/ibi.12054

Hargitai, R., Boross, N., Nyiri, Z. et al. Biliverdin- and protoporphyrin-based eggshell pigmentation in relation to antioxidant supplementation, female characteristics and egg traits in the canary (Serinus canaria). Behav Ecol Sociobiol 70, 2093–2110 (2016). https://doi.org/10.1007/s00265-016-2214-z

Hargitai, R. Nóra Boross, Zoltán Nyiri, Zsuzsanna Eke. Effects of food limitation on the intensity of blue-green and brown eggshell coloration: an experimental study with the canary. Journal of Avian Biology Volume 49, Issue 1 jav-01486 First published: 15 September 2017 https://doi.org/10.1111/jav.01486

Jianfei Chen, Seyed Benyamin Dalirsefat, Deping Han, Xianggui Dong, Guoying Hua, Xiaotong Zheng, Tianlan Xia, Tianqi Shao, Xuemei Deng, Changxin Wu, An EAV-HP insertion in the 5ʹ flanking region of SLCO1B3 is associated with its tissue-expression profile in blue-eggshell Yimeng chickens (Gallus gallus), Poultry Science, Volume 99, Issue 12, 2020, Pages 6371-6377, ISSN 0032-5791, https://doi.org/10.1016/j.psj.2020.09.002.

Ming-Yuan Lu, Wei-Wei Wang, Guang-Hai Qi, Li Xu, Jing Wang, Mitochondrial transcription factor A induces the declined mitochondrial biogenesis correlative with depigmentation of brown eggshell in aged laying hens, Poultry Science, Volume 100, Issue 3, 2021,100811, ISSN 0032-5791, https://doi.org/10.1016/j.psj.2020.10.065.

Morales, J., Velando, A. & Torres, R. Biliverdin-based egg coloration is enhanced by carotenoid supplementation. Behav Ecol Sociobiol 65, 197–203 (2011). https://doi.org/10.1007/s00265-010-1025-x

Moreno, J. Elisa Lobato, Judith Morales. Eggshell blue-green coloration fades immediately after oviposition: a cautionary note about measuring natural egg colors. Ornis Fennica 88 (1), 51, 2011.

Navarro, C. Tomás Pérez-Contreras, Jesús M. Avilés, Kevin J. McGraw, Juan J. Soler. Journal of Avian BiologyVolume 42, Issue 6 p. 538-543. Blue-green eggshell coloration reflects yolk antioxidant content in spotless starlings Sturnus unicolor. First published: 30 November 2011 https://doi.org/10.1111/j.1600-048X.2011.05293.x

Ostertag, E. Miriam Scholz, Julia Klein, Karsten Rebner, Dieter Oelkrug. Pigmentation of white, brown, and green chicken eggshells analyzed by reflectance, transmittance, and fluorescence spectroscopy. Chemistry  1084, 2019.

Samiullah, S. J.R. Roberts, The location of protoporphyrin in the eggshell of brown-shelled eggs, Poultry Science, Volume 92, Issue 10, 2013, Pages 2783-2788, ISSN 0032-5791, https://doi.org/10.3382/ps.2013-03051.

Samiullah, S. J.R. Roberts, K. Chousalkar, Eggshell color in brown-egg laying hens — a review, Poultry Science, Volume 94, Issue 10, 2015, Pages 2566-2575, ISSN 0032-5791, https://doi.org/10.3382/ps/pev202.

Sparks, N. Eggshell Pigments–from Formation to Deposition. December 2011.

Wang Z, Qu L, Yao J, Yang X, Li G, et al. (2013) An EAV-HP Insertion in 5′ Flanking Region of SLCO1B3 Causes Blue Eggshell in the Chicken. PLOS Genetics 9(1): e1003183. https://doi.org/10.1371/journal.pgen.1003183

Wang, Z., Meng, G., Bai, Y. et al. Comparative transcriptome analysis provides clues to molecular mechanisms underlying blue-green eggshell color in the Jinding duck (Anas platyrhynchos). BMC Genomics 18, 725 (2017). https://doi.org/10.1186/s12864-017-4135-2

Wang, Z. R.F. Liu, A.R. Wang, J.Y. Li, X.M. Deng, Expression and activity analysis reveal that heme oxygenase (decycling) 1 is associated with blue egg formation, Poultry Science, Volume 90, Issue 4, 2011, Pages 836-841, ISSN 0032-5791,https://doi.org/10.3382/ps.2010-01143.

WANG, ZhePeng; LIU, RuiFang and WANG, AnRu. Comparison of HMOX1 expression and enzyme activity in blue-shelled chickens and brown-shelled chickens. Genet. Mol. Biol.[online]. 2013, vol. 36, n. 2, pp. 282-286. ISSN 1415-4757. https://doi. org/10.1590/S1415-47572013000200020.

Wiemann, J., Yang, TR. & Norell, M.A. Reply to: Egg pigmentation probably has an Archosaurian origin. Nature570, E46–E50 (2019). https://doi.org/10.1038/s41586-019-1283-3

Wragg D, Mwacharo JM, Alcalde JA, Wang C, Han J-L, Gongora J, et al. (2013) Endogenous Retrovirus EAV-HP Linked to Blue Egg Phenotype in Mapuche Fowl. doi:10.1371/journal.pone.0071393
« Last Edit: June 10, 2021, 06:03:29 PM by Lindsay Helton »
Joshua 24:15

Lindsay Helton

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This study was a good read and alludes to two new pigments that have been identified which affect egg color. It will be neat to see what discoveries the scientific community makes regarding eggshell color overtime.

https://www.researchgate.net/publication/342813890_Expanding_the_eggshell_colour_gamut_uroerythrin_and_bilirubin_from_tinamou_Tinamidae_eggshells

“Despite the diversity of observable colors, there is a universal consensus that all of these colors are generated by only two pigments‬: the tetrapyrrolic compounds protoporphyrin IX and ‬biliverdin IX. Only the blue-green pigment biliverdin has been previously detected in the eggshells of the Spotted Nothura and the Elegant Crested‬ Tinamou. ‭Tinamou eggs exhibit a diversity of bright colors. and suggest the presence of other ‭pigments. We thus hypothesized that the eggshells are generated by mixing biliverdin with other, yet unknown, ‬pigments. We examined these tinamou eggshells, specifically extracting and identifying their pigments, and analysed their contributions to the observed eggshell coloration.‬ This study discovered the orange pigment uroerythrin ‭3‭H‬ and the yellow–brown pigment bilirubin ‭4‭H‬‬. We ‬can confidentially conclude that both of these newly found pigments are genuine eggshell pigments as we experi‭mentally verified that they are not artifacts generated in the extraction process.”
« Last Edit: June 06, 2021, 03:40:52 PM by Lindsay Helton »
Joshua 24:15

Lindsay Helton

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This scientific study offers a great explanation for why blue egg color tends to fade over the course of the laying cycle.

https://scholar.google.com/scholar?hl=en&as_sdt=0%2C43&q=blue+eggshell+gene+modification&oq=blue+eggshell+gene+modi#d=gs_qabs&u=%23p%3Dn79NVtxTCJMJ

“The formation mechanism underlying the blue eggshell characteristic has been discovered in birds, and SLCO1B3 is the key gene that regulates the blue eggshell color. Insertion of an endogenous retrovirus, EAV-HP, in the 5’ flanking region of the SLCO1B3 gene promotes the expression of SLCO1B3 in the chicken shell gland. However, at different laying stages of the same group of chickens, the color of the eggshell can vary widely, and the molecular mechanism underlying the eggshell color change remains unknown. We analyzed the change in the eggshell color during the laying period. The results indicated that the eggshell color in Lushi chickens can be divided into 3 stages: 20-25 weeks for dark blue, 26-45 weeks for medium blue, and 46-60 weeks for light blue. We further investigated the expression and methylation levels of the SLCO1B3 gene at 8 different weeks, and the results showed that the relative expression level of the SLCO1B3 gene was significantly higher at 25 and 30 weeks than at other laying weeks. Furthermore, the overall methylation rate of the SLCO1B3 gene in Lushi chickens increased gradually with increasing weeks of egg production, as shown by bisulfite sequencing PCR. Pearson correlation analysis showed that methylation of the promoter region of SLCO1B3 was significantly negatively correlated with both SLCO1B3 expression in the shell gland tissue and eggshell color. In addition, we predicted that CpG5 and CpG8 may be key sites for regulating SLCO1B3 gene transcription. Our findings show that as the level of methylation increases, methylation of the CpG5 and CpG8 sites hinders the binding of transcription factors to the promoter, reducing the expression of SLCO1B3 during the late period and resulting in a lighter eggshell color.”
Joshua 24:15