Effects of different energy levels in low-protein diet on liver lipid metabolism in the late-phase laying hens through the gut-liver axis

Abstract

Background: The energy/protein imbalance in a low-protein diet induces lipid metabolism disorders in late-phase laying hens. Reducing energy levels in the low-protein diet to adjust the energy-to-protein ratio may improve fat deposition, but this also decreases the laying performance of hens. This study investigated the mechanism by which different energy levels in the low-protein diet influences liver lipid metabolism in late-phase laying hens through the enterohepatic axis to guide feed optimization and nutrition strategies. A total of 288 laying hens were randomly allocated to the normal-energy and normal-protein diet group (positive control: CK) or 1 of 3 groups: low-energy and low-protein diet (LL), normal-energy and low-protein diet (NL), and high-energy and low-protein diet (HL) groups. The energy-to-protein ratios of the CK, LL, NL, and HL diets were 0.67, 0.74, 0.77, and 0.80, respectively.

Results: Compared with the CK group, egg quality deteriorated with increasing energy intake in late-phase laying hens fed low-protein diet. Hens fed LL, NL, and HL diets had significantly higher triglyceride, total cholesterol, acetyl-CoA carboxylase, and fatty acid synthase levels, but significantly lower hepatic lipase levels compared with the CK group. Liver transcriptome sequencing revealed that genes involved in fatty acid beta-oxidation (ACOX1, HADHA, EHHADH, and ACAA1) were downregulated, whereas genes related to fatty acid synthesis (SCD, FASN, and ACACA) were upregulated in LL group compared with the CK group. Comparison of the cecal microbiome showed that in hens fed an LL diet, Lactobacillus and Desulfovibrio were enriched, whereas riboflavin metabolism was suppressed. Cecal metabolites that were most significantly affected by the LL diet included several vitamins, such as riboflavin (vitamin B₂), pantethine (vitamin B₅ derivative), pyridoxine (vitamin B₆), and 4-pyridoxic acid.

Conclusion: A lipid metabolism disorder due to deficiencies of vitamin B₂ and pantethine originating from the metabolism of the cecal microbiome may be the underlying reason for fat accumulation in the liver of late-phase laying hens fed an LL diet. Based on the present study, we propose that targeting vitamin B₂ and pantethine (vitamin B₅ derivative) might be an effective strategy for improving lipid metabolism in late-phase laying hens fed a low-protein diet.

Keywords: Cecal microbiome; Energy/protein imbalance; Late-phase laying hens laying hens; Liver lipid metabolism; Low-protein diet; Multi-omics.

Fig. 1

Differences of fat level indice in the liver of aged laying hens among different groups. Different lowercase letters in each box of the same sub-figure represent significant differences among aged laying hens from different groups (Tukey’s HSD test, P < 0.05)

Fig. 2

Variations in the activities of lipid metabolic enzymes in the liver of aged laying hens among different groups. Different lowercase letters in each box of the same sub-figure represent significant differences among aged laying hens from different groups (Tukey’s HSD test, P < 0.05)

Fig. 3

Liver transcriptome of aged laying hens fed an LP diet. a PCA exhibiting the variations in the gene expression profiles. b Volcano plot showing the results of DEG identification. c KEGG annotation of DEGs. d KEGG enrichment analysis of DEGs. e The PPI network of DEGs

Fig. 4

Cecal metabolites of aged laying hens in the CK and LL groups. a OPLS-DA revealing the variations in cecal metabolites. b Volcano plot showing the results of DAM identification. c VIP plot exhibiting the DAMs with the strongest variation between the CK and LL groups. d KEGG pathway enrichment analysis of DAMs

Fig. 5

Variations in the composition of the cecal microbiome induced by the LP diet. a PCoA and the adonis test based on the unweighted and weighted UniFrac distances revealing the differences in cecal microbiome composition between the CK and LL groups. b Bacterial phyla and genera with significant variation in relative abundance in the cecum between the CK and LL groups (Student’s t-test, P < 0.05). c SPEC-OCCU plots showing ASVs in the cecal microbiome of aged laying hens fed a normal or LP diet

Fig. 6

Co-occurrence network and predicted function of the cecal microbiome. a Co-occurrence networks of the cecal microbiome from the CK and LL samples. Nodes belonging to different modules are labeled in different colors. b Zi-Pi plot showing the distribution of bacterial ASVs based on their topological roles. c Robustness and vulnerability of networks of the LL and CK groups. Different lowercase letters above the bars of the robustness plot represent a significant difference between the CK and LL groups (Student’s t-test, P < 0.05). d PCoA and adonis test based on the Bray–Curtis distance revealing the differences in cecal microbiome functions related to human disease and organismal systems between the CK and LL groups

Fig. 7

Correlation analyses among liver lipid metabolism indices, liver DEGs, cecum DAMs, different cecal microbiome functions, and different cecal bacteria, and the conceptual frameworks of deduced mechanisms of lipid metabolism disorder in aged laying hens fed the LL diet