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The eggshell colour is related to the biological functions of birds, and the colour of poultry eggshells can affect consumers' choices. This study explored the difference in the metabolite composition of duck eggshells to screen the key substances that affect the eggshell colours. The green and white duck eggshells were selected for non-targeted metabolomics analysis. We screened 402 and 512 differentially expressed metabolites in the negative and positive ion modes, respectively. Among them, 40 differentially expressed metabolites were annotated with specific names and related functions, of which the expression levels of 8 metabolites showed extremely significant differences. They were 2-heptanone, 12-hydroxydodecanoic acid, D-fructose, dodecanedioic acid, L-leucine, methyl jasmonate, palmitoleic acid, and styrene oxide. Additionally, the annotated differentially expressed metabolites were enriched in 33 metabolic pathways, including aminoacyl-tRNA biosynthesis, amino acid metabolism, galactose metabolism, etc. The results showed that the expression of metabolites between green and white eggshells differed in ducks; among metabolites with extremely significant differences in expression, the expression level of L-leucine in green eggshells was higher than that in ducks white eggshells. Therefore, we speculated that the increased expression of L-leucine promoted the response of related metabolic pathways, enhanced the expression of antioxidants, and changed the eggshell colours.HighlightsThe metabolites of green and white shells were different.40 DEMs were annotated with specific names and related functions.The annotated DEMs were enriched in 33 metabolic pathways. The metabolites of green and white shells were different. 40 DEMs were annotated with specific names and related functions. The annotated DEMs were enriched in 33 metabolic pathways.

Metabonomics reveals the main small molecules differences between green and white egg shells in ducks