Metabolomic analysis of the egg yolk during the embryonic development of broilers

Abstract

The chicken egg yolk, which is abundant with lipids, proteins, and minerals, is the major nutrient resource for the embryonic development. In fact, the magnitude and type of yolk nutrients are dynamically changed during the chicken embryogenesis to meet the developmental and nutritional requests at different stages. The yolk nutrients are metabolized and absorbed by the yolk sac membrane and then used by the embryo or other extraembryonic tissues. Thus, understanding the metabolites in the yolk helps to unveil the developmental nutritional requirements for the chicken embryo. In this study, we performed ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) analysis to investigate the change of metabolites in the egg yolk at embryonic (E) 07, E09, E11, E15, E17, and E19. The results showed that 1) the egg yolk metabolites at E07 and E09 were approximately similar, but E09, E11, E15, E17, and E19 were different from each other, indicating the developmental and metabolic change of the egg yolk; and 2) most of the metabolites were annotated in amino acid metabolism pathways from E11 to E15 and E17 to E19. Especially, arginine, lysine, cysteine, and histidine were continuously increased during the embryonic development, probably because of their effects on the growth promotion and oxidative stress amelioration of the embryo. Interestingly, the ferroptosis was found as one of major processes occurred from E15 to E17 and E17 to E19. Owing to the upregulated expression of acyl-CoA synthetase long-chain family member 4 detected in the yolk sac, we assumed that the ferroptosis of the yolk sac was perhaps caused by the accumulation of reactive oxygen species, which was induced by the large amount of polyunsaturated fatty acids and influx of iron in the yolk. Our findings might offer a novel understanding of embryonic nutrition of broilers according to the developmental changes of metabolites in the egg yolk and may provide new ideas to improve the health and nutrition for prehatch broiler chickens.

Keywords: chicken; embryogenesis; metabolomics; yolk.

Figure 1

The principal component analysis (PCA) and sparse partial least squares discriminant analysis (sPLSDA) plots of the metabolites that were significantly changed during the chicken embryogenesis. Each plot was generated from the z-scored data of metabolite abundances that were significantly changed during the embryogenesis. The 2 major components that accounted for the most variation of the metabolite abundance were used to plot. Each dot in the figure represented each sample, and different colors indicated the embryonic stage. (A) Positive mode data, PCA plot. (B) Positive mode data, sPLSDA plot. (C) Negative mode data, PCA plot. (D) Negative mode data, sPLSDA plot. (E) Combined data, PCA plot. (F) Combined data, sPLSDA plot.

Figure 2

Major KEGG pathways enriched at different embryonic stages. KEGG pathways were searched based on the differential metabolites identified from each pair of embryonic days, such as E7-E9 means that the differential metabolites were calculated and enriched for KEGG pathway analysis when compared between E7 and E9. The cutoff (red dotted line) was selected as P < 0.1. The y axis means the -LOG (P value). Different colors of the bars represent the different pair group of embryonic days.

Figure 3

Relative fold change of amino acid content in the chicken egg yolk during the embryonic development. Each section presents the different amino acid subfamily that is defined by the structure or function, such as α-ketoglutarate subfamily (A), oxaloacetate subfamily (B), glycerol-3-phosphate subfamily (C), branch-chained amino acid subfamily (D), aromatic amino acid subfamily (E), and histidine subfamily (F). The fold change (y axis) was calculated by dividing the abundance of each amino acid at E07, and the abundance of each amino acids in egg yolk is displayed in Table 2.

Figure 4

The fold change of critical metabolites in urea cycle and arginine-creatine pathway in the egg yolk during the embryogenesis. (A) Change of arginine and citrulline. (B) Change of arginine and its downstream metabolites (creatine, creatinine and sarcosine).

Figure 5

The fold change of lysine and carnitine in the egg yolk during the embryogenesis.

Figure 6

Relative gene expression of GPX4 and ACSL4 in chicken yolk sac at different embryonic days. The error bar represents standard deviation (SD) and the significance was represented as ∗∗∗ (P < 0.001).