Effect of a Carotenoid Extract from Citrus reticulata By-Products on the Immune-Oxidative Status of Broilers

Abstract

Although carotenoids generally possess antimicrobial and antioxidant properties, the in vivo synergistic action of carotenoid blends derived from plant-based by-products has not been thoroughly studied. Therefore, the carotenoid characterization and antimicrobial potential of Citrus reticulata extract as well as the impact of this carotenoid-rich extract (CCE) dietary supplementation on the performance, meat quality, and immune-oxidative status of broiler chickens were determined. One hundred and twenty one-day-old hatched chicks (Ross 308) were allocated to two dietary groups, with four replicate pens of 15 birds each. Birds were fed either a basal diet (CON) or the basal diet supplemented with 0.1% CCE (25 mg carotenoid extract included in 1 g of soluble starch) for 42 d. β-Cryptoxanthin, β-Carotene, Zeaxanthin, and Lutein were the prevailing carotenoid compounds in the Citrus reticulata extract. The CCE feed additive exerted inhibitory properties against both Gram-positive (Staphylococcus aureus) and negative (Klebsiella oxytoca, Escherichia coli, and Salmonella typhimurium) bacteria. Both the broiler performance and meat quality did not substantially differ, while the breast muscle malondialdehyde (MDA) concentration tended to decrease (p = 0.070) in the CCE-fed broilers. The inclusion of CCE decreased the alanine aminotransferase and MDA concentration, and the activity of glutathione peroxidase, while the activity of superoxide dismutase was increased in the blood. Catalase and NADPH oxidase 2 relative transcript levels were significantly downregulated in the livers of the CCE-fed broilers. Additionally, Interleukin 1β and tumor necrosis factor (TNF) relative transcript levels were downregulated in the livers of the CCE- fed broilers, while TNF and interferon γ (IFNG) tended to decrease in the spleens and bursa of Fabricius, respectively. The present study provided new insights regarding the beneficial properties of carotenoids contained in Citrus reticulata in broilers' immune-oxidative status. These promising outcomes could be the basis for further research under field conditions.

Keywords: Liquid Chromatography-Mass Spectrometry (LC/MS-MS); antimicrobials; antioxidants; bursa of Fabricius; immune; liver; meat; spleen.

Figure 1

The effect of Citrus reticulata carotenoid extract included in soluble starch in the relative viability of E. coli, K. oxytoca, S. typhimurium, and S. aureus using MTT assay.

Figure 2

The total antioxidant capacity and lipid peroxidation index of breast muscle of broilers fed the two diets (Control; CON and carotenoid extract from Citrus reticulata included in soluble starch; CCE) at 42 days. FRAP: Ferric Reducing Ability of Plasma, ABTS: 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate), DPPH: 2,2′-diphenyl-1-picrylhydrazyl radical, and MDA: Malondialdehyde.

Figure 3

The mean and standard error of means (SEM) of selective hematological and biochemical parameters in blood serum of broilers fed the two experimental diets (Control; CON and carotenoid extract from Citrus reticulata included in soluble starch; CCE) at 42 days. SGOT-AST: aspartate aminotransferase, SGPT-ALT: alanine aminotransferase, BUN: blood urea nitrogen, γ-GT: γ-glutamyltransferase, SAP: alkaline phosphatase, CHOL: cholesterol, and CP: total proteins and fractions of albumins (ALB) and globulins (GLOB).

Figure 4

The mean and standard error of means (SEM) of relative transcript levels as fold changes of several genes involved in the antioxidant system in the liver of broilers fed the two experimental diets (Control; CON and carotenoid extract from Citrus reticulata included in soluble starch; CCE) at 42 days. SOD: Superoxide Dismutase, CAT: Catalase, GPX1: Glutathione Peroxidase 1, GPX2: Glutathione Peroxidase 2, NOS2: Nitic Oxide Synthase 2, NOX1: NADPH oxidase 1, NOX2: NADPH oxidase 2, NOX3: NADPH oxidase 3, and GSTA2: Glutathione Transferase A2.

Figure 5

The mean ± SEM of total antioxidant capacity, oxidative stress indicators, and enzyme activities (Units/mL), in the blood plasma of broilers fed the two experimental diets (Control; CON, and carotenoid extract from Citrus reticulata included in soluble starch; CCE) at 42 days. SOD: Superoxide Dismutase, CAT: Catalase, GST: Glutathione Transferase, GSH-Px: Glutathione Peroxidase, GR: Glutathione Reductase, FRAP: Ferric Reducing Ability of Plasma, ABTS: 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate), DPPH: 2,2′-diphenyl-1-picrylhydrazyl radical, and MDA: Malondialdehyde.

Figure 6

The mean and standard error of means (SEM) of relative transcript levels as fold changes of several genes involved in the immune system in the liver of broilers fed the two experimental diets (Control; CON and carotenoid extract from Citrus reticulata included in soluble starch; CCE) at 42 days. INFA: Translation initiation factor IF-1, INFG: Interferon-gamma, IL18: Interleukin 18, IL1B: Interleukin 1 Beta, IL2: Interleukin 2, IL8: C-X-C Motif Chemokine Ligand 8, MAPK: Mitogen-activated protein kinase, NFKB: Nuclear factor-kappa B, TLR4: Toll-like receptors 4, and TNF: Tumor necrosis factor.

Figure 7

The mean and standard error of means (SEM) of relative transcript levels as fold changes of several genes involved in the immune system in the spleen of broilers fed the two experimental diets (Control; CON and carotenoid extract from Citrus reticulata included in soluble starch; CCE) at 42 days. INFA: Translation initiation factor IF-1, INFG: Interferon-gamma, IL18: Interleukin 18, IL1B: Interleukin 1 Beta, IL2: Interleukin 2, IL8: C-X-C Motif Chemokine Ligand 8, IL6: Interleukin 6, MAPK: Mitogen-activated protein kinase, NFKB: Nuclear factor-kappa B, TLR4: Toll-like receptors 4, and TNF: Tumor necrosis factor.

Figure 8

The mean and standard error of means (SEM) of relative transcript levels as fold changes of several genes involved in the immune system in the bursa of Fabricius of broilers fed the two experimental diets (Control; CON and carotenoid extract from Citrus reticulata included in soluble starch; CCE) at 42 days. INFA: Translation initiation factor IF-1, INFG: Interferon-gamma, IL18: Interleukin 18, IL1B: Interleukin 1 Beta, IL2: Interleukin 2, IL8: C-X-C Motif Chemokine Ligand 8, IL6: Interleukin 6, MAPK: Mitogen-activated protein kinase, NFKB: Nuclear factor-kappa B, TLR4: Toll-like receptors 4, and TNF: Tumor necrosis factor.