Comparative Study of an Antioxidant Compound and Ethoxyquin on Feed Oxidative Stability and on Performance, Antioxidant Capacity, and Intestinal Health in Starter Broiler Chickens

Abstract

Concerns over the safety of ethoxyquin (EQ) highlight the need for safer, more effective feed antioxidants. This study investigated a healthier antioxidant compound (AC) as a potential alternative to EQ in broilers. A total of 351 one-day-old Arbor Acres Plus male broilers were randomly assigned to three treatments for 21 days: control (CON), EQ group (200 g/ton EQ at 60% purity), and AC group (200 g/ton AC containing 18% butylated hydroxytoluene, 3% citric acid, and 1% tertiary butylhydroquinone). AC supplementation reduced the acid value, peroxide value, and malondialdehyde content in stored feed, decreased feed intake and the feed conversion ratio without affecting body weight gain, and enhanced antioxidant capacity (liver total antioxidant capacity and superoxide dismutase; intestinal catalase and glutathione peroxidase 7). It improved intestinal morphology and decreased barrier permeability (lower diamine oxidase and D-lactate), potentially by promoting ZO-1, Occludin, and Mucin2 expression. The AC also upregulated NF-κB p50 and its inhibitor (NF-κB p105), enhancing immune regulation. Additionally, the AC tended to increase beneficial gut microbiota, including Lactobacillus, and reduced Bacteroides, Corprococcus, and Anaeroplasma. Compared to EQ, the AC further enhanced feed oxidative stability, the feed conversion ratio, intestinal morphology and barrier functions, and inflammatory status, suggesting its potential as a superior alternative to EQ for broiler diets.

Keywords: antioxidant capacity; antioxidant compound; broiler; ethoxyquin; intestinal health.

Figure 1

Oxidative stability of feeds after three and six weeks of storage. N = 3. (A) Acid value (AV); (B) peroxide value (POV); (C) malondialdehyde (MDA) content. CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. ^abc Different superscript letters indicate significant differences (p < 0.05).

Figure 2

Growth performance of broilers. N = 9. (A) Body weight gain (BWG); (B) feed intake (FI); (C) feed conversion ratio (FCR). CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. ^ab Different superscript letters indicate significant differences (p < 0.05).

Figure 3

Liver antioxidant capacity of broilers. N = 8. (A) Total antioxidant capacity (T-AOC); (B) superoxide (SOD) activity; (C) catalase (CAT) activity; (D) glutathione peroxidase (GSH-Px) activity; (E) reduced glutathione (GSH) content; (F) malondialdehyde (MDA) content. CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. ^ab Different superscript letters indicate significant differences (p < 0.05).

Figure 4

Intestinal barrier permeability and intestinal-barrier-related protein expression. N = 8. (A) Serum diamine oxidase activity and (B) D-lactate contents. (C–F) The relative protein expression of ZO-1, Occludin, and MUC2 in the jejunum. CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. Abbreviations: DAO, diamine oxidase. ^ab Different superscript letters indicate significant differences (p < 0.05).

Figure 5

Intestinal-antioxidant-related protein expression. N = 8. (A–D) Relative protein levels of kelch-like ECH-associated protein 1 (KEAP1), nuclear factor erythroid 2-related factor 2 (NRF2), and catalase (CAT) in jejunum. (E–H) Relative protein levels of heme oxygenase-1 (HO-1), glutathione peroxidase 7 (GPX7), and NAD(P)H quinone dehydrogenase 1 (NQO1) in jejunum. N = 8. CON, basal diet; CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. ^ab Different superscript letters indicate significant differences (p < 0.05).

Figure 6

Expression of inflammatory cytokines at gene level and protein level. N = 8. (A) Relative mRNA expression of IL-1β, IL-18, IL-6, IFN-γ, and TNF-α in jejunum. (B–F) Relative protein levels of NF-κB p50, NF-κB p105, inhibitor of nuclear factor kappa-B (IκB), and toll-like receptor 4 (TLR4) in jejunum. CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. ^ab Different superscript letters indicate significant differences (p < 0.05).

Figure 7

Changes in microbial diversity in the cecum of broilers. N = 6. (A) Venn diagram of the operational taxonomic units (OTUs) among all groups. (B) Bacterial richness (Chao1 and Observed species) and diversity (Shannon and Simpson) were evaluated by Kruskal–Wallis rank sum test and Dunn’s test. (C) The rarefaction curve shows the Chao1 index of each group under the same sampling depth. (D) The principal coordinate analysis (PCoA) and (E) non-metric multidimensional scaling (NMDS) analysis were conducted at the ASV level (the distance matrix was analyzed for statistical significance using PERMANOVA). CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton compound antioxidant.

Figure 8

Changes in microbial community composition in the cecum of broilers. N = 6. (A) Distribution of cecal microbiota at the phylum level. (B) Heatmap showing the relative abundance of cecal microbiota at the phylum level. (C–E) Bacteria with differences in relative abundance among the top 10 phyla, including Firmicutes, Bacteroidetes, and the ratio of Bacteroidetes and Firmicutes. (F) Distribution of cecal microbiota at the genus level. (G) Heatmap showing the relative abundance of cecal microbiota at the genus level. (H–K) Bacteria with differences in relative abundance among the top 20 genera, including Lactobacillus, Bacteroides, Coprococcus, and Anaeroplasma. Differences in microbial abundance between the CON and AC groups were analyzed using the Wilcoxon rank sum test. CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound.

Figure 9

Graphic summary of the effects of the antioxidant compound on feed antioxidant protection and broiler health. Antioxidant compound: including 18% butylated hydroxytoluene, 3% citric acid, and 1% tertiary butylhydroquinone.