The pigments in eggshell with different colour and the pigment regulatory gene expression in corresponding chicken's shell gland.

Eggshell colour is the unique appearance and economically valuable trait of eggs, whereas the colour is often short of uniformity, especially in the blue-shelled breeds, hence, their pigment differences and molecular mechanism need clarity. To investigate the relationship between the pigment content of eggshells and related gene expression in the eggshell glands of chickens, four subtypes of blue-shelled eggs ('Olive', 'Green', 'Blue', and 'Light') from the same blue-eggshell chicken line were selected; Hy-Line 'White' and 'Brown'-shelled eggs were used as control groups. The L*, a*, b* values, and protoporphyrin-IX and biliverdin contents in each group of eggshells were measured. In addition, the shell glands of the corresponding hens were collected to detect SLCO1B3 genotype and mRNA expression, and ABCG2 and HMOX1 transcription and protein expression. Eggshell colour L* values were negatively correlated with protoporphyrin-IX, b* values were positively correlated with total pigment content (P < 0.001), and a* values were positively correlated with protoporphyrin-IX (P < 0.001) but negatively with biliverdin. Moreover, all four blue-eggshell subtypes were SLCO1B3 homozygous, with SLCO1B3 mRNA expression in shell glands being significantly higher than in the White and Brown groups. ABCG2 and HMOX1 mRNA expression were highest in the Brown and Green groups, respectively (P < 0.05), and were positively correlated with protoporphyrin-IX (P < 0.001) and biliverdin contents in eggshells, respectively. Western blot and immunohistochemical results demonstrated that the Brown group had the highest ABCG2 expression (P < 0.05), followed by the Green and Olive groups. HMOX1 protein expression was higher in the Olive and Green groups (P < 0.05), and lowest in the White group. This study suggests that ABCG2 and HMOX1 have important regulatory roles in the production and transport of protoporphyrin-IX and biliverdin in blue-shelled chicken eggs, respectively.