Dietary Organic Zinc Supplementation Modifies the Oxidative Genes via RORγ and Epigenetic Regulations in the Ileum of Broiler Chickens Exposed to High-Temperature Stress

Abstract

Heat stress (HS) is a significant concern in broiler chickens, which is vital for global meat supply in the dynamic field of poultry farming. The impact of heat stress on the ileum and its influence on the redox homeostatic genes in chickens remains unclear. We hypothesized that adding zinc to the feed of heat-stressed broilers would improve their resilience to heat stress. However, this study aimed to explore the effects of organic zinc supplementation under HS conditions on broiler chickens' intestinal histology and regulation of HS index genes. In this study, 512 Xueshan chickens were divided into four groups: vehicle, HS, 60 mg/kg zinc, and HS + 60 mg/kg zinc groups. Findings revealed that zinc supply positively increased the VH and VH: CD in the ileum of the broilers compared to the HS group, while CD and VW decreased in Zn and HS+Zn supplemented broilers. Zn administration significantly increased superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and decreased the enzymatic activities of reactive oxygen species (ROS) and malondialdehyde (MDA) compared to the HS group. In addition, Zn administration significantly increased relative ATP, complex I, III, and V enzyme activity compared to the HS group. Furthermore, the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), lactate transporter 3 (LPCAT3), peroxiredoxin (PRX), and transferrin receptor (TFRC) in the protein levels was extremely downregulated in HS+Zn compared to the HS group. Zn supply significantly decreased the enrichment of RORγ, P300, and SRC1 at target loci of ACSL4, LPCAT3, and PRX compared to the HS group. The occupancies of histone active marks H3K9ac, H3K18ac, H3K27ac, H3K4me1, and H3K18bhb at the locus of ACSL4 and LPCAT3 were significantly decreased in HS+Zn compared to the HS group. Moreover, H3K9la and H3K18la at the locus of ACSL4 and LPCAT3 were significantly decreased in HS+Zn compared to the HS group. This study emphasizes that organic Zn is a potential strategy for modulating the oxidative genes ACSL4, LPCAT3, PRX, and TFRC in the ileum of chickens via nuclear receptor RORγ regulation and histone modifications.

Keywords: RORγ; epigenetics; ileum; oxidative stress; zinc.

Figure 1

This figure shows the effects of Zn supplementation on the growth performance of the heat-stressed chickens. (A) Body weight. (B) Average daily feed intake. Data are presented as means ± SD. * p < 0.05.

Figure 2

This figure shows the effects of Zn supplementation on the ileum histomorphology of the heat-stressed chickens. (A) Villus height (VH). (B) Crypt depth (CD). (C) Villus height/ Crypt depth (VH/CD). (D) Villus width (VW). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, ° = the number of replication.

Figure 3

Effects of Zn supplementation on the oxidative stress of the heat-stressed chickens. (A) Superoxide dismutase (SOD). (B) Catalase (CAT). (C) Glutathione peroxidase (GSH). (D) Malondialdehyde (MDA). (E) Reactive oxygen species (ROS). (F–I) The relative parameters of mitochondria ATP, enzyme complex-I, complex-III, and complex-V. Data are presented as means ± SD. * p < 0.05, ° = the number of replication.

Figure 4

Effects of Zn supplementation on the ileum expressions of genes included in antioxidation of the heat-stressed chickens. (A) mRNA expression changes of the antioxidants related. (B,C) protein levels changes of the antioxidants related. Data are presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, ° = the number of replication.

Figure 5

Effects of Zn supplementation on the ileum transcriptional activation of gene expression of the heat-stressed chickens. (A) The interactions among RORγ, SRC, PPARα, and core proteins involved in cholesterol metabolism during transcriptional regulation were predicted by Search Tool for the Retrieval of Interacting Genes (STRING). (B,C) Western blotting analysis was performed to evaluate the expression of nuclear RORγ, P300, SRC, PPARα, at the protein level. Data are presented as means ± SD. ** p < 0.01, *** p < 0.001, ° = the number of replication.

Figure 6

Effects of Zn supplementation on the ileum, the enrichment of RORγ, P300, and SRC1 at target loci of ACSL4, LPCAT3, PXR, and TFRC genes and their physical interaction of the heat-stressed chickens. (A–D) ChIP-qPCR analyses of RORγ, P300, and SRC1 occupancies at the locus of ACSL4, LPCAT3, PXR, and TFRC. (E,F) The relative enrichment of coactivator RNA polymerase II, RNA polymerase II-SER2, and RNA polymerase II-SER5 at the locus of ACSL4 and LPCAT3. Data are presented as means ± SD. * p < 0.05, *** p < 0.001, ° = the number of replication.

Figure 7

The effects of Zn supplementation on the ileum, histone modification at the locus of ACSL4 and LPCAT3 in heat-stressed chickens. (A–I) The relative enrichment of histone marks’ (H3K9ac, H3K118ac, H3K27ac, H3K4me1, H3K9bhb, H3K18bhb, H3K9la, H3K18la, and H4K8la) occupancy was analyzed by ChIP-qPCR. Data are presented as means ± SD. * p < 0.05, *** p < 0.001, ° = the number of replication.