Comparison of protoporphyrin IX content and related gene expression in the tissues of chickens laying brown-shelled eggs

Abstract

Protoporphyrin IX (PpIX), an immediate precursor of heme, is the main pigment resulting in the brown coloration of eggshell. The brownness and uniformity of the eggshell are important marketing considerations. In this study, 9 chickens laying darker brown shelled eggs and 9 chickens laying lighter brown shelled eggs were selected from 464 individually caged layers in a Rhode Island Red pureline. The PpIX contents were measured with a Microplate Reader at the wavelength of 412 nm and were compared in different tissues of the 2 groups. Although no significant difference in serum, bile, and excreta was found between the 2 groups, PpIX content in the shell gland and eggshell of the darker group was higher than in those of the lighter group, suggesting that PpIX was synthesized in the shell gland. We further determined the expression levels of 8 genes encoding enzymes involved in the heme synthesis and transport in the liver and shell gland at 6 h postoviposition by quantitative PCR. The results showed that expression of aminolevulinic acid synthase-1 (ALAS1) was higher in the liver of hens laying darker brown shelled eggs, whereas in the shell gland the expression levels of ALAS1, coproporphyrinogen oxidase (CPOX), ATP-binding cassette family members ABCB7 and ABCG2, and receptor for feline leukemia virus, subgroup C (FLVCR) were significantly higher in the hens laying darker brown shelled eggs. Our results demonstrated that hens laying darker brown shelled eggs could deposit more PpIX onto the eggshell and the brownness of the eggshell was dependent on the total quantity of PpIX in the eggshell. More heme was synthesized in the liver and shell gland of hens laying darker brown shelled eggs than those of hens laying lighter brown shelled eggs. High expression level of ABCG2 might facilitate the accumulation of PpIX in the shell gland.

iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness

“…The L* value was used to represent the intensity of eggshell brownness, as verified by the high correlation coefficient between the L*value and protoporphyrin IX quantity in the eggshell [11]. Lower L* values are associated with darker eggshells, and vice-versa.…”

“…The intensity of eggshell brownness is dependent on the concentration of protoporphyrin IX in the eggshell [11], which will be affected by the total amount of protoporphyrin IX and the size of eggshell. Given the same amount of protoporphyrin IX, larger eggs will have lighter brownness than smaller eggs.…”

“…At 6 h post-oviposition the next egg reaches the shell gland to undergo mineralization during nearly 19 hours; meanwhile, the protoporphyrin IX granules can be observed in the epithelium of the shell gland, suggesting that the proteins associated with protoporphyrin IX synthesis and transport are active at this time. Our previous studies indicated that the intensity of eggshell brownness was determined by the content of protoporphyrin IX deposited onto the eggshell, and that the protoporphyrin IX content in the eggshell depends on the accumulation of protoporphyrin IX in the shell gland epithelium [11]. Therefore, the differentially expressed proteins (S2 Table) that are involved in protoporphyrin IX synthesis and transport warrant further investigation.…”

“…Thus, eggs with protoporphyrin IX deposited onto the shell exterior will appear brown. Protoporphyrin IX destined for the avian eggshell is synthesized in the shell gland [8–11] and accumulates in the epithelial cells progressively after ovulation. It is secreted into the uterine fluid in the final stage of eggshell formation; therefore, protoporphyrin IX is mainly found in the cuticle of the eggshell [12].…”

A selection study for improving eggshell colour in two parent lines of laying hens and their hybrids

“…The SCI was deemed suitable for reliable colour identification only in brown and non-pigmented eggs. Research by Li et al (2013) reported that a comparison of protoporphyrin IX in different tissues, of light-and deep-brown egg-laying RIR hens, indicated that eggshell pigment was synthesised in the shell gland. Moreover, there was a significantly higher gene expression of d-aminolevulinic acid synthase recorded in the deep-brown egg-laying hens as compared with hens laying lighter coloured eggs.…”