Abstract
Green-shelled eggs are widely recognized for their high quality, nutritional value, and superior shell strength, making them highly attractive to consumers. In ducks, the trait for green shells is dominant, while the trait for white shells is recessive. Notably, even within homozygous dominant populations, considerable variation in eggshell color intensity remains, affecting product uniformity and market value. In this study, homozygous green-shelled laying ducks at 30 and 70 weeks of age into three groups based on shell color measurements: dark-green, medium-green, and light-green. The content of biliverdin decreased significantly with age and exhibited considerable variation among the three-color groups, while protoporphyrin IX content showed no consistent trend or significant differences. A highly significant positive correlation was found between the thickness of the shell gland epithelium and biliverdin content in in ducks at 30 weeks, which remained significant at 70 weeks. Furthermore, at 70 weeks, shell color correlated positively with biliverdin content in the shell gland, where both biliverdin and biliverdin IX beta levels were significantly higher than those found in the liver. Transcriptomic analysis revealed that ST13 gene expression decreased with age, concurrent with lightening of shell color. Metabolomic profiling showed that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) contents in the shell gland increased with darker shell color and were significantly positively correlated with ST13 expression. Weighted Gene Co-expression Network Analysis (WGCNA) suggested that ST13 is co-expressed with ALAS1 and may be involved in regulating porphyrin metabolism, as well as the metabolic pathways of glycine, serine, and threonine.