Effects of Different Levels of Ethanolic Propolis Extract and Multi-Strain Probiotic on Performance, Immune Response, Gut Microflora, and Antioxidant Status in Broiler Chickens

Document Type : Research Articles

Authors

Ehsan Salehifar

Ali Asghar Nikoukar

Said Sobhanirad

Department of Agricultural Science, Ma. C., Islamic Azad University, Mashhad, Iran

10.22067/ijasr.2025.94890.1266

Abstract

Introduction: The increasing global demand for antibiotic-free poultry products has accelerated research into natural alternatives that can enhance growth performance, health, and welfare of broiler chickens. Among these alternatives, propolis a resinous substance produced by honeybees is rich in bioactive compounds such as flavonoids, phenolic acids, and aromatic esters, which exhibit antimicrobial, antioxidant, and immunomodulatory properties. Probiotics are also widely used for their beneficial effects on gut microbiota modulation, nutrient absorption, and immune function. Therefore, this study aimed to evaluate the effects of different dietary levels of ethanolic propolis extract (300, 600, and 900 mg/kg), compared with a commercial probiotic, on growth performance, humoral immune response, cecal microbial populations, and antioxidant status in broiler chickens under standard rearing conditions.
Materials and Methods: A total of 240 one-day-old Cobb 500 broiler chicks were allocated to five dietary treatments, with four replicates per treatment and 15 birds per replicate. The experimental treatments consisted of: (1) a control diet without additives; (2) the control diet supplemented with 300 mg/kg ethanolic propolis extract; (3) the control diet supplemented with 600 mg/kg ethanolic propolis extract; (4) the control diet supplemented with 900 mg/kg ethanolic propolis extract; and (5) the control diet supplemented with a commercial probiotic.Birds were reared for 42 days with ad libitum access to feed and water. The experimental diets were formulated to meet nutrient requirements according to the Ross 500 guidelines. Growth performance parameters, including daily weight gain (DWG), feed intake (FI), and feed conversion ratio (FCR), were recorded during starter, grower, finisher, and overall periods. At the end of the experiment, cecal contents were aseptically collected to enumerate Lactobacillus spp. and Escherichia coli using selective culture media. Blood samples were collected from the wing vein to determine serum immunoglobulin G (IgG) and total immunoglobulin (Total Ig) concentrations using a hemagglutination assay. Antioxidant status was evaluated by measuring serum activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD), as well as malondialdehyde (MDA) concentration, a marker of lipid peroxidation and oxidative stress. Data were analyzed by ANOVA, and treatment means were compared using the appropriate post hoc test when significant differences were detected. Statistical significance was declared at P < 0.05.
Results and Discussion: Dietary supplementation improved overall daily weight gain compared with the control group, with the highest DWG observed in birds receiving 900 mg/kg propolis (P < 0.05). Across production phases, the 900 mg/kg dose increased FI during the grower and finisher periods (P≤0.005), consistent with enhanced appetite and/or palatability. Despite higher feed intake at the highest inclusion level, the overall feed conversion ratio (FCR) decreased in the supplemented groups (P=0.002), indicating improved feed efficiency at the flock level. These findings suggest that propolis and the probiotic can support growth by enhancing digestive and absorptive functions, although dose–response patterns may diverge between growth stimulation (pronounced at 900 mg/kg) and feed efficiency (often optimized at moderate inclusion). Both propolis and probiotic treatments increased Lactobacillus counts and decreased Escherichia coli (P<0.05). The dual pattern expansion of beneficial lactic acid bacteria alongside suppression of E. coli supports the concept that these additives can stabilize gut ecology in favor of commensal populations. Such modulation is mechanistically compatible with the observed performance benefits, given the recognized roles of Lactobacillus in competitive exclusion, pH reduction via organic acid production, and enhancement of epithelial barrier function. Propolis supplementation decreased MDA (P<0.05; reported P=0.02), consistent with lower lipid peroxidation. In parallel, activities of GPx and SOD increased in the supplemented groups (P<0.05; GPx P=0.04; SOD P=0.002), pointing to strengthened endogenous antioxidant defenses. This biochemical profile plausibly contributes to improved nutrient utilization and growth by mitigating oxidative stress in rapidly growing broilers. No significant differences were detected among treatments for IgG or total immunoglobulins (P>0.05). While null effects on these humoral markers were observed under the present conditions, the performance and antioxidant advantages indicate that propolis and probiotics may act primarily through gut and metabolic pathways rather than by amplifying systemic antibody titers in non-challenged birds. Collectively, the integrated outcomes (performance, microbiota, and antioxidant indices) point to complementary mechanisms microbiota modulation and redox homeostasis as proximate drivers of the observed benefits.
Conclusions: Dietary supplementation with ethanolic propolis extract and a multi-strain probiotic improved several key outcomes in broiler chickens. Overall, daily weight gain increased with supplementation, with the 900 mg/kg propolis level yielding the highest growth, while the feed conversion ratio over the entire experimental period decreased in the supplemented groups. Cecal Lactobacillus counts increased, whereas Escherichia coli populations decreased (P < 0.05), indicating a healthier intestinal microbial balance. Antioxidant capacity improved, as evidenced by decreased malondialdehyde (MDA) concenterations and increased activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD). Humoral immune indices (including serum IgG and total immunoglobulin levels, were not significantly affected. These findings position propolis and probiotics as practical, natural additives to support growth performance, gut health, and oxidative resilience. Further work should delineate dose optimization and the molecular pathways underlying redox and microbiome shifts, and assess long-term impacts under diverse rearing conditions or pathogenic challenges.

Keywords

Antioxidant enzyme

Cecal microbiota

Feed additive

Humoral immunity

Oxidative stress

Subjects

• Poultry Nutrition

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Authors retain the copyright. This is an open access article distributed under Creative Commons Attribution 4.0 International License (CC BY 4.0)

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