Abstract
The laying hen's industry faces significant ethical and economic challenges due to day-old male chick culling, driving the need for reliable in ovo sexing methods. While chicken embryo allantoic fluid (AF) shows promise for early sex determination through sex-specific biomarkers, current research lacks comprehensive sex-specific analyses of fertilized egg AF. Moreover, AF production and metabolism involve dynamically complex, multi-component regulatory interactions that require further investigation. To address this knowledge gap, we systematically investigated the sex-specific molecular characterization of egg yolk and AF from day 1 and day 12 of chicken embryo development using metabolomics and proteomics techniques. Egg yolk proteomics analysis showed that the metabolic requirements of chicken embryos for yolk-derived nutrients increased progressively as embryonic development progressed. It was found that although sex-specific protein expression patterns persisted throughout the developmental stages, the degree of expression differences gradually diminished with developmental time, suggesting that the protein expression of male and female embryos showed convergence in the later stages of development. Metabolomic profiling of yolk demonstrated a significant expansion of sex-specific metabolic differences by late embryonic development (day 12), with activation of steroidogenic pathways consistent with established sexual differentiation mechanisms. Metabolomic analysis of AF revealed sexually dimorphic features, mainly in the differential expression of key metabolic pathways such as nucleotide biosynthesis, energy partitioning and signal transduction. Compared with yolk components at the same developmental stage, AF metabolic profiles demonstrated stronger sex differentiation. Weighted gene co-expression network analysis based on 1183 AF metabolites identified a sex-specific co-regulatory module (Pearson r = 0.57, p = 0.05) that was significantly enriched in ectodermal membrane development (male-biased) and energy metabolism pathways, and revealed a synergistic regulatory relationship between AF and yolk metabolic components. This study provides a multi-omics resource characterizing yolk and AF dynamics during chicken embryogenesis. Our findings elucidate sex-specific metabolic divergence and phenotypic outcomes between male and female embryos, advancing understanding of avian developmental biology and the mechanistic basis of sexual dimorphism in chicken embryos.