Integrative analysis of metabolomics and transcriptomics reveals alterations in egg quality and hepatic lipid metabolism in hens supplemented with curcumin

Abstract

Curcumin has been shown to affect lipid metabolism in various ways, but its molecular mechanisms in hens remain poorly understood. In this study, 378 Hy-Line Brown hens, aged 58 weeks, were randomly assigned into three groups, each consisting of 6 replicates with 21 hens per replicate. The control group was fed a basal diet, while the experimental groups received diets supplemented with either 50 or 100 mg/kg of curcumin for 42 days. The results indicated that 50 mg/kg curcumin supplementation significantly increased average egg weight (quadratically, P = 0.001) and decreased the feed conversion ratio (FCR) (quadratically, P = 0.018). Both 50 and 100 mg/kg curcumin supplementation increased the yolk weight (linearly, P = 0.010), yolk color score (linearly, P = 0.001), and thick albumin weight (quadratically, P = 0.003), thereby improving egg quality. Additionally, curcumin supplementation at both doses reduced hepatic triglyceride (TG) content (linearly, P = 0.007) and hepatic free cholesterol (FC) content (linearly, P = 0.003), while increasing serum catalase (CAT) activity (linearly, P = 0.008) and hepatic superoxide dismutase (SOD) activity (linearly, P = 0.013), alleviating hepatic steatosis and oxidative stress in laying hens. Transcriptomic analysis revealed that curcumin primarily upregulated key genes involved in hepatic cholesterol synthesis (Fdft1, Sqle, Cyp51A1, Msmo1, and Dhcr24) and downregulated key genes involved in hepatic TG synthesis (Acaca, Acacb, and Fasn). Metabolomic analysis identified upregulated serum lysoPS 18:1 as the most significant explanatory variable in curcumin-fed hens. Integrated metabolomics and transcriptomics further revealed that lysoPS 18:1 positively correlated with hepatic cholesterol synthesis genes (Fdft1, Sqle, Cyp51A1, and Msmo1) and negatively correlated with hepatic TG synthesis gene (Fasn), suggesting its role in reducing hepatic TG and FC metabolism. In conclusion, these findings indicate that curcumin enhances production performance and egg quality of laying hens, improves hepatic lipid metabolism, and that 50 mg/kg is the optimal supplementation dose.

Keywords: Curcumin; Laying hen; Lipid metabolism; Metabolomics; Transcriptomics.

Fig. 1

Improvement of hepatic lipid deposition through curcumin. (A) Representative gross morphology of the liver after 6 weeks of feeding in the Con, LC, and HC groups of laying hens. (B) Representative liver sections with H&E staining. (C) Representative liver sections with Oil red O staining. (D) Histological scores of liver sections. Data are represented as mean ± SEM (n = 4). Different lowercase letters in the same row indicate significant differences (P < 0.05). CON, control; LC, 50 mg/kg curcumin supplementation; HC, 100 mg/kg curcumin supplementation. H&E = hematoxylin and eosin.

Fig. 2

Overview of hepatic transcriptome analysis in laying hens. (A) Volcano plot of compounds detected in Con and Cur groups. Thresholds were | Log ₂ (fold change) | > 1 and P < 0.05. (B) Bar plot of DEGs. (C) 3D PCA plot of DEGs. (D) Top 20 KEGG enrichment pathways. (E) The heatmap-bar plot of DEGs from significant pathways. (F) mRNA levels of significant genes in laying hens. (G) Linear regression of mRNA expression and FPKM of genes in fatty acid biosynthesis pathway. (H) Linear regression of mRNA expression and FPKM of genes in cholesterol biosynthesis pathway. CON = control; Cur = curcumin; FC = fold change; DEGs = differentially expressed genes; KEGG = Kyoto Encyclopedia of Genes and Genomes; FPKM = fragments per kilobase million. ∗, P < 0.05; ∗∗, P < 0.01.

Fig. 3

Hepatic expression pathways of laying hens. (A) Pathway diagram of cholesterol biosynthesis-related gene regulation. (B) Pathway diagram of de novo lipogenesis-related gene regulation. VLDL = very low-density lipoprotein cholesterol; HMG-CoA = 3-hydroxy-3-methylglutaryl-coenzyme A; TG = triglyceride; TC = total cholesterol; FC = free cholesterol; CE = cholesterol ester.

Fig. 4

Serum metabolite changes in laying hens. (A) Bar plot of MS2 metabolites. (B–C) PLS-DA plot of the Con and Cur groups. (D) Volcano plot of DAMs. Thresholds were | Log ₂ (fold change) | > 1 and P < 0.05. (E) Pie chart of KEGG class of DAMs. (F) Heatmap plot of DAMs. (G) The expression level change (Z-scored original value) of upregulated metabolites. (H) The expression level change (Z-scored original value) of downregulated metabolites. Asterisks indicate statistical significance based on unpaired two-sided Student's t-test. P value: ∗, <0.05; ∗∗, <0.01; ∗∗∗, <0.001. MS2 = tandem mass spectrometry; PLS-DA = partial least squares discriminant analysis; DAMs = differentially accumulated metabolites; CON = control; Cur = curcumin.

Fig. 5

Integrated analysis of transcriptomic and metabolomic. (A) Mantel's correlation analysis. Pairwise correlations between metabolites were shown, with a color gradient denoting Pearson's correlation coefficient. Egg quality metrics (based on the egg quality test in week 6), liver health metrics (based on the serum biochemical test and liver TG content test), and DEGs were related to DAMs by partial Mantel's tests. The solid line represents a positive correlation, and the dashed line represents a negative correlation. Edge width and color denote the statistical significance based on 9,999 permutations. The edge with P < 0.05 has been removed. (B) Nine-quadrant map of DAMs and DEGs. (C) Correlation network of DAMs and DEGs. (D) Correlation network of DAMs, egg quality metrics, and liver health metrics. (E) Sankey diagram visualizing relationships between DEGs, DAMs, and production metrics (egg quality and liver health). TG = triglyceride; DEGs = differentially expressed genes; DAMs = differentially accumulated metabolites.