A Review of the Blue Eggshell Pigments of Ameraucana Fowl
by Lindsay Helton
The blue eggshell of an Ameraucana fowl is one of the attributes that draws people to the Ameraucana breed. In this post, the biochemical and physiological characteristics of the blue egg shell are reviewed. Numerous studies and scientific advances have occurred over the years which have gradually increased our knowledge of the blue-green pigments that create the characteristic blue egg shell of an Ameraucana fowl. It is important to note that the pigment has been renamed several times over the course of the past century, which may lead to some confusion if you research the topic. In the 1800s, and in several studies completed since then, the pigment that causes blue eggs is referred to as biliverdin. In the late 1800s, H.C. Sorby completed a scientific study and referred to the same pigment as Oocyan (Sorby, 1875). As scientific advances have occurred, more in-depth studies on eggshell pigments have been completed. The blue-green pigments that create blue eggshells are presently identified as biliverdin-IX and zinc biliverdin chelate.
Friedrich Tiedemann (1781-1861), a german physiologist and anatomist, was one of the first individuals to study the blue-green pigment that we now refer to as biliverdin. He created the now famous gmelin reaction (a diagnostic color test) which was able to identify bilirubin in bile. Bilirubin is a derivative of biliverdin (Tiedemann, 1814).
In 1858, W. Wicke presented what is considered to be the first known chemical essay on egg color pigments. Wicke treated egg shells with hydrochloric acid, water and boiling alcohol. During the process, he collected and identified a bluish green pigment that he declared to be biliverdin (Wicke, 1858).
In 1875, H.C. Sorby published "Proceedings of the Zoological Study," which is considered to be the first scientifically based publication on the pigments present in blue eggs. He completed spectrum analysis of colored egg shells and identified seven substances present in various egg shells. Those substances were labeled: 1) Oorhodeine, 2) Oocyan, 3) Banded Oocyan, 4) Yellow Ooxanthine, 5) Rufous Ooxanthine, 6) Lichnoxanthine, and 7) a substance imperfectly distinguished (Sorby, 1875). Oocyan ("oo" = egg, "cyan" = blue) eventually became known as biliverdin within the scientific community. The pigment Sorby labeled as Oorhodeine eventually became known as Protoporphyrin IX. Protoporphyrin IX has been identified as the pigment that creates brown egg shell color.
R.C. Punnett was the first individual to classify the pigment found on brown egg shells as protoporphyrin. Punnett also discovered that the blue-green eggshell color in chickens was produced by an autosomal dominant gene called Oocyan. Punnet noted in his scientific study that eggs laid by Oocyan homozygotes were a darker blue than those laid by heterozygotes (Punnett, 1933).
Kennedy and Vevers completed a study in 1973 and found that blue eggs laid by Araucanas contained biliverdin-IX, zinc biliverdin chelate, and protoporphyrin-IX (Kennedy, et al., 1973). They completed an additional study on 108 Araucana fowl in 1976 and confirmed that only the pigments biliverdin-IX and zinc biliverdin chelate could be detected in blue eggs and were causal of the blue coloring. They confirmed in their studies that brown egg shells contained large amounts of the pigment protoporphyrin-IX (Kennedy, et al., 1976). The following is an excerpt of the findings they made during the study:
“Eggshells from 108 species were examined for appearance and pigment content. Principal pigments found were protoporphyrin, biliverdin IXα and its zinc chelate. 49 species had protoporphyrin only, 2 had biliverdin only, 33 had protoporphyrin and biliverdin, 17 had all 3 pigments, one species had biliverdin and its zinc chelate, one had protoporphyrin and biliverdin zinc chelate and five species had no pigment. Biliverdin zinc chelate was never found alone (Kennedy et al., 1976).”
In pullets and hens that lay an egg on the olive green or khaki spectrum, a coating of the pigment protoporphyrin-IX has been added to the egg during the final hours of the egg being inside the uterus. Biliverdin, the pigment responsible for blue egg color, passes through all eight layers of the egg shell. If you crack open a blue Ameraucana egg shell, you will find that the interior of the egg shell is also blue (Wang et al., 2007).
The higher the concentration of biliverdin within the uterus of a hen, the bluer the egg will be. The higher the concentration of protoporphyrin-IX within the uterus of a hen, the more olive or brown an egg will be (Wang et al., 2009). This information was confirmed in the study below.
“The quantity of biliverdin of Dongxiang blue-shelled chickens was much more than Dongxiang brown-shelled chickens, whereas the quantity of protoporphyrin of Dongxiang blue-shelled chickens was only about half that of Dongxiang brown-shelled chickens, which hinted at the probable different transformation from precursor to biliverdin or protoporphyrin between blue-shelled chickens and brown-shelled chickens (Wang et al., 2009).”
If you click the link below, there is a table in the published study that lists the pigments that were studied and the correlation between the amount of each pigment and the egg shell color produced.
https://academic.oup.com/ps/article/88/8/1735/2886225 Interestingly, scientific research has shown that biliverdin is created within the shell gland and then is deposited on the eggshell roughly 3-4 hours before ovi-position (Wang, et al., 2009).
A study was done in 2010 to map the blue egg allele locus. The blue egg allele is identified by the letter “O.” A total of 98 blue egg laying hens were studied, and the map was identified as (TTA) n –(TG) n –A–O–(tg) n. The study also found that the “O locus was located between theA and (tg) nloci, that is, Chr1:67,296,991-69,140,571, which is the first genomic sequence interval to be established for the blue eggshell gene (Wang et al., 2010).”
A research study completed in 2013 found that a blue egg occurs due to the expression of the SLCO1B3 gene. The SLCO1B3 gene is a part of the organic anion transporting polypeptide (OATP) family. The OATPs function as membrane transporters, and have been proven to transport bile products such as Biliverdin. An EAV-HP insertion in the 5′ flanking region of the SLCO1B3 gene was found to be connected to the blue egg phenotype (Wang et al., 2013).
Many folks are unaware that the color varieties of Ameraucanas were initially created by crossing birds of varying backgrounds and selecting for specific attributes. Blue egg color was one part of that equation. The attributes that were selected for were eventually used to create the breed standard for each of the approved Ameraucana color varieties. The APA standard calls for Ameraucanas to have several other important attributes as well, such as a pea comb, reddish bay eye color, a beard, muffs, slate shanks, etc. It is important to remember that there are no perfect birds out there. Breeding birds towards the standard of perfection is all about balance. Don't become so lost in any one feature or attribute that you forget about the other aspects of the standard.
Narrow your birds down carefully each season and keep the ones that most closely adhere to the standard. If you need to improve egg color in your Ameraucana flock, toe punch chicks that hatch from the bluest eggs. If the chicks grow up to be pullets, you can monitor them and hopefully they will lay an equally blue egg. If the chicks grow up to be cockerels, you will have foreknowledge that they hatched from a very nicely colored blue egg and are most likely carrying good blue egg genes. Good luck with your Ameraucana flock!
I included a photo of an Ameraucana egg shell below to show how the biliverdin-IX and zinc biliverdin chelate pigments pass through the entire egg shell.
REFERENCES
Kennedy, G. Y., and H. G. Vevers. 1976. A survey of avian eggshell pigments. Comp. Biochem. Physiol. 55B:117–123
Kennedy, G. Y., and H. G. Vevers. 1973. Eggshell pigments of the Araucano fowl. Comp. Biochem. Physiol. 44B:11–25.
Punnett, C. 1933. Genetic Studies in Poultry. J. Genet. 27:465-470.
Sorby, H. C. (1875). On the colouring-matters of the shells of birds' eggs. Proceedings of the Zoological Society of London 1875: 351–365
Tiedemann, F. Journal de Pharmacie, Tome XXIII, p. 109.
Wang Z, Qu L, Yao J, Yang X, Li G, Zhang Y, et al. (2013) An EAV-HPInsertion in 5′ Flanking Region of SLCO1B3 Causes Blue Eggshell in the Chicken. PLoS Genet 9(1): e1003183.
https://doi.org/10.1371/journal.pgen.1003183 Wang, X.T., C. J. Zhao, J. Y. Li, G. Y. Xu, L. S. Lian, C. X. Wu, X. M. Deng, Comparison of the total amount of eggshell pigments in Dongxiang brown-shelled eggs and Dongxiang blue-shelled eggs, Poultry Science, Volume 88, Issue 8, August 2009, Pages 1735–1739,https://doi.org/10.3382/ps.2008-00434
Wang, X.T., J.-R. Bai, C.-J. Zhao, H. Zhang, H.-G. Bao, G.-Y. Xu, J.-Y. Li, Dr L.-S. Lian, C.-X. Wu & Dr X.-M. Deng (2010) Localisation of the genomic sequence interval for the blue eggshell gene using an F2 resource population of Dongxiang chickens, British Poultry Science, 51:4, 507-509, DOI: 10.1080/00071668.2010.502520
Wang, X.T., Deng, C.-J. Zhao, J.-Y. Li, G.-Y. Xu, L.-S. Lian, C.-X. Wu, Study of the Deposition Process of Eggshell Pigments Using an Improved Dissolution Method, Poultry Science, Volume 86, Issue 10, October 2007, Pages 2236–2238,
https://doi.org/10.1093/ps/86.10.2236 Wicke, W. (1858). Uber des pigment in den eischalen der vogel. Naumannta 8: 393–397.
Zhao, R., G.-Y. Xu, Z.-Z. Liu, J.-Y. Li, N. Yang, A study on eggshell pigmentation: biliverdin in blue-shelled chickens, Poultry Science, Volume 85, Issue 3, March 2006, Pages 546–549,
https://doi.org/10.1093/ps/85.3.546
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