Abstract:

【Background】 Brown eggshell color is closely relevant to eggshell strength, concentrations of egg white antimicrobial protein and yolk carotenoid, blood and meat spots, and hatchability, and is an important index affecting quality and sale of eggs. However, due to absence of selection for eggshell color, color of brown eggs that some indigenous breeds lay is light and highly variable, which has an adverse effect on sale of eggs and creation of egg brands. 【Objective】 The aims of this study are to establish a quantization method that could accurately and sensitively capture the variation of eggshell color, to estimate breed-specific heritability of eggshell color and to identify candidate genes associated with eggshell color in the Lüeyang black-boned chicken (LBC), so as to provide a theoretic basis for genetic improvement of LBC eggshell color. 【Method】 841 hens from 62 half-sibling families of LBC breeding population were selected. Three eggs were collected from each hen, and then the eggshell color was quantified using the L*a*b color space, and posterior heritabilities of L, a, and b values were estimated in a Bayesian framework using the Markov Chain Monte Carlo (MCMC) algorithm. The prior distributions of breeding value and residual variances were set using an inverse-Gamma distribution. MCMC performed 130 000 iterations, dropped 30 000 iterations at the beginning and stored one every 100 iterations to obtain posterior distributions of variance components and posterior heritability estimates of color indexes. Hens that laid light brown (n=8), brown (n=8) and light blue (n=8) eggs were selected from LBC population. Three eggs were collected from each hen for the measurement of eggshell pigment, and the absorbance values were measured at 670 nm for biliverdin and at 412 nm for protoporphyrin. The concentrations of protoporphyrin and biliverdin were calculated by regression equations, which were fitted using absorbances and concentrations of standard samples. Shell glands of brown- (n=8) and light brown-shelled (n=8) hens were collected. Expression levels of ALAS1 and CPOX in shell glands were detected using qPCR. 【Result】 Among three color indexes, a* value kept a high consistency with the change of eggshell color based on subjective perception as a* value generally decreased with transition of egg color from brown to green hues. L and b values could accurately reflect the change of egg color from light to dark brown, but L and b values were unable to discriminate between brown and green hues reliably. L and b values showed low variation as L and b values of 50% of samples were distributed between 73.3-79.1 and 13.1-18.2. In contrast, a* values were evenly distributed among the samples as a* values of 50% of samples ranged from 1.4 to 7.1. In line with the distribution characteristics, the coefficient of variation (88.2%) of a value was higher than ones of L (5.7%) and b (24.0%) values. Estimation results of heritability showed that a* value (h²=0.77) was predominately affected by genetic factors. In contrast, L (h²=0.46) and b (h²=0.37) values were controlled by environmental effect to larger extents. For the relationship of eggshell pigment and color, protoporphyrin concentration had a strong correlation with all of three color indexes (L: r=-0.86, a: r=0.73, b: r=0.88). But biliverdin concentration showed a strong (r=-0.73) negative correlation with a* value alone. ALAS1 and CPOX were two key enzymes that catalyze the biosynthensis of protoporphyrin precursors. Expression results showed that expression levels of CPOX in shell glands of brown-shelled chickens were 1.5-fold higher (P<0.05) than that in shell glands of light brown-shelled chickens. ALAS1 had no significant (P>0.05) difference between brown- and light brown-shelled chickens. 【Conclusion】 These results indicated that protoporphyrin was the key pigment affecting LBC eggshell color, and CPOX was a candidate associated with protoporphyrin concentration and color of brown eggs. The a* value was an optimal index quantifying eggshell color of LBC with high accuracy and sensitivity. In view of high heritability estimate of a* value, it was possible to increase brown hue and uniformity of LBC eggshell color via positive selection of a* value.

Key words: Lüeyang black-boned chicken, eggshell color, L*a*b color space, heritability, CPOX, ALAS1