Effect of Spirulina supplementation on immune and antioxidant parameters of commercial broilers

Introduction

Poultry nutrition is undergoing a significant transition, shaped by evolving production demands and increasing scrutiny of conventional feeding practices. Historically, synthetic antimicrobials and chemical feed additives were widely incorporated into intensive poultry diets to promote rapid growth, improve feed efficiency, and provide effective disease prevention. However, rising concerns regarding antimicrobial resistance, consumer safety, and sustainability have driven the search for safer and more natural nutritional alternatives. In this context, growing research evidence supports the inclusion of naturally derived bioactive substances such as phytochemicals, essential oils, organic acids, probiotics, and microalgae in poultry feeding programs. These compounds exhibit multiple functional roles, contributing to enhanced growth performance, improved nutrient utilization, strengthened immune responses, increased antioxidant defense, and better intestinal health. Among these alternatives, microalgae have emerged as promising candidates for future-oriented poultry nutrition strategies (Rafiq et al., 2022) [15] . One microalga of particular interest is Arthrospira platensis, commonly known as Spirulina, a filamentous cyanobacterium belonging to the genus Arthrospira, which also comprises A. maxima and A. fusiformis. Spirulina is recognized for its exceptionally high protein content, richness in essential vitamins, and abundance of bioactive pigments such as phycocyanin with strong antioxidant properties (Ogbuewu et al., 2025) [11] . Due to these attributes, Spirulina has attracted considerable attention as a functional feed ingredient in poultry diets. Previous studies have demonstrated that its dietary inclusion in broilers can positively influence meat quality, nutritional composition, sensory characteristics, and the accumulation of health-promoting bioactive compounds (Toyomizu et al., 2001) [16] . The functional value of Spirulina is further enhanced by its diverse phenolic profile, including salicylic, trans-cinnamic, sinapic, chlorogenic, quinic, and caffeic acids, which collectively contribute to its pronounced antioxidant activity (Lassiter et al., 2024) [8] . Additionally, its rapid growth rate and relatively uncomplicated cultivation process have facilitated its commercial-scale production in several regions across the globe. Despite the extensive documentation of Spirulina's nutritional richness and biofunctional properties, research focused on its optimal dietary inclusion and targeted effects in broiler chickens remains limited and fragmented. Hence, the present study was undertaken to evaluate the influence of Spirulina supplementation on growth performance and gut health in broiler chickens, with the aim of generating evidence to support its effective utilization under commercial poultry production conditions.

Materials and Methods

Three hundred (300) day old broiler chicks were randomly allocated to five dietary treatment groups with twelve (12) replicates of five (5) birds each and all the experimental groups were reared under unvarying uniform managemental conditions. The birds were vaccinated against RD with a live vaccine of La Sota on 7th and 28th day and against IBD on 14th and 21st day of the experiment. Corn and Soyabean meal based basal diet was formulated for birds to meet the nutrient requirements during pre-starter, starter and finisher phases. Treatment groups included were T1 as control which is basal diet without any feed additive, T2 with antibiotic (Lincomycin-Positive control) @ 100 g/MT diet, T3 with (Spirulina platensis) @ 0.5%, T4 with (Spirulina platensis) @ 0.75% and T5 with (Spirulina platensis) @ 1.0%. Broiler pre-starter, starter and finisher diets were formulated to contain 22%, 20.5% and 19% crude protein and metabolizable energy was 3000, 3100 and 3200 kcal/kg respectively.

Immune Parameters 2.1.1 Cell mediated Immune response

Cell-mediated immune response was evaluated using the cutaneous basophilic hypersensitivity (CBH) reaction to phytohaemagglutinin-phosphate (PHA-P). On the 40th day of the experimental period, one bird was randomly selected from each replicate for the assessment. The thickness of the skin web between the third and fourth digits of both the left and right feet was measured using a micrometer prior to injection. The right foot web was inoculated intradermally with 100 µg of PHA-P dissolved in 0.1 ml of phosphatebuffered saline (PBS), whereas the left foot web served as the control and received 0.1 ml of PBS alone. After 24 hours, the thickness of both webs was again measured with a micrometer, and the CBH response was calculated using the standard formula.

Antioxidant response 2.2.1 Glutathione Peroxidase (GSHPx) Enzyme Activity in Serum

Glutathione peroxidase (GSH-Px) activity was determined according to the method described by Paglia and Valentine (1967), with minor modifications for microplate analysis [13] . The assay was performed in 96-well microtiter plates. Each reaction well contained 12.5 µl of serum, 250 µl of 0.1 mM phosphate-buffered saline (PBS; pH 7.4), 12.5 µl of hydrogen peroxide (H₂O₂), and 12.5 µl of reduced glutathione. The reaction mixture was allowed to incubate at room temperature for 5 minutes. Subsequently, 12.5 µl of nicotinamide adenine dinucleotide phosphate (NADPH) was added to initiate the enzymatic reaction. The decrease in absorbance was monitored at 340 nm using an ELISA microplate reader for a duration of 5 minutes, with readings recorded at 60-second intervals. Enzyme activity was expressed as units per milligram of protein.

Superoxide Dismutase (SOD) Enzyme Assay in Serum

Serum superoxide dismutase (SOD) activity was determined using a microplate-based assay following the procedure described by Madesh and Balasubramanian (1998) [9] . The assay was carried out in 96-well microtiter plates, wherein 100 µl of serum sample was dispensed into each well. This was followed by the addition of 6 µl of 1.25 mM 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution, 15 µl of freshly prepared 100 µM pyrogallol, and 29 µl of 25 mM phosphate-buffered saline (PBS), resulting in a final reaction volume of 150 µl per well. Pyrogallol was prepared immediately prior to use and added after all other reagents had been introduced. The reaction mixture was incubated at room temperature for 10 minutes. Thereafter, 150 µl of dimethyl sulfoxide (DMSO) was added to each well to terminate the reaction and solubilize the MTT formazan crystals. The plates were shaken thoroughly, and absorbance was recorded at 570 nm using an ELISA microplate reader (BioTek Instruments µQuant).

Lipid Peroxidation in Serum

Lipid peroxidation in serum was estimated by measuring malondialdehyde (MDA) levels using the thiobarbituric acid reactive substances (TBARS) method as described by Ohkawa et al. (1979) [12] . In this assay, MDA formed during lipid peroxidation reacts with thiobarbituric acid (TBA) under acidic and high-temperature conditions to produce a pink-colored chromogen, which is subsequently extracted into butan-1-ol. The intensity of the resulting chromogen was measured spectrophotometrically to quantify lipid peroxide concentration. Results were expressed as nanomoles of MDA per milligram of protein. For the assay, 100 µl of serum was transferred into a clean, dry test tube, followed by the addition of 100 µl of distilled water and 50 µl of 8% (w/v) sodium dodecyl sulfate. The mixture was allowed to stand at room temperature for 10 minutes. Thereafter, 375 µl each of 20% acetic acid and TBA reagent were added, and the samples were heated in a boiling water bath for 60 minutes to facilitate color development. After cooling to room temperature, 2.5 ml of distilled water and 1.25 ml of a butanol-pyridine mixture (15:1, v/v) were added, followed by thorough mixing. The absorbance of the organic phase was recorded at 542 nm using a spectrophotometer. To minimize interference from residual chromogenic components and improve photometric clarity, alkaline pyridine-butanol was used to achieve a clear solution suitable for direct absorbance measurement.

Statistical analysis

The data was analyzed using the General Linear Model procedure of Statistical Package for Social Sciences (SPSS) 20th version and comparison of means was done using Duncan's multiple range test and significance was considered at p < 0.05.

Results and Discussion

Immune Parameters

Different dietary supplementations did not show significant (p > 0.05) effect on the cell mediated immune response to Phytohemagglutinin-P (PHA-P). Khan et al. (2020) reported that Spirulina at 1.0 g/kg (SPA1), 1.5 g/kg (SPA2), numerically improved lymphoproliferative skin response compared with the control group (SPA0), whereas birds fed SPA2 and SPA3 showed the greatest increase in skin thickness after 24 hr, confirming enhanced CMI in SPA3 but non-significant (p > 0.05) [7] . Contrary to the present finding, Khadanga et al. (2023) demonstrated significant improvements in cellular immunity with Spirulina at 0.5-1.5%, including increased CBH response and increased lymphoid organ weights [6] . The immunomodulatory effects of Spirulina are attributed to bioactive compounds including phycocyanin, β-carotene, polysaccharides and essential fatty acids, which stimulate macrophage activity, enhance natural killer cell function and promote antibody production. The lack of significant effects in the present study's cell-mediated immunity assessment may be attributed to differences in Spirulina dosage, duration of supplementation, or the specific immune challenge protocol employed.

Serum Antioxidant Enzyme Responses 3.2.1 Glutathione Peroxidase (GPx)

The concentration of glutathione peroxidase differed significantly (p < 0.05) among the dietary treatments. Birds fed Spirulina (1.0%) recorded the highest GPx activity, which was significantly higher than the values observed in the T1 and T2 groups. The GPx activity in T3 and T4 remained intermediate and did not differ significantly from either T5 or the lower-performing groups. The lowest GPx concentration was recorded in the control (T1) group. These findings are in agreement with Abdelfatah et al. (2024) who reported that Spirulina supplementation at 0.1%, 0.3% and 0.5% significantly enhanced GPx activity, with 0.5% inclusion producing the strongest antioxidant response [1] . Park et al. (2018) observed that dietary Spirulina up to 1.0% significantly elevated serum GPx activities, reflecting enhanced systemic antioxidant capacity [14] . However, in contrast Ahmed et al. (2022) findings did not show any significance at 3% and 5% [3] .

Lipid Peroxidation (MDA)

Lipid peroxidation levels, expressed as nmol MDA/mg protein, were significantly (p < 0.05) reduced in birds receiving Spirulina supplementation compared with the control group. The lowest MDA concentration was observed in T5, followed by T4, both of which differed significantly from the control (T1). The T2 and T3 groups showed intermediate values that did not differ significantly from the higher Spirulina fed groups. The reduction in lipid peroxidation is crucial for maintaining cell membrane integrity, protecting cellular components from oxidative damage and supporting overall metabolic efficiency. Moustafa et al. (2021) reported significant enhancements in antioxidant status under cyclic heat stress, with Spirulina reducing MDA and increasing antioxidant capacity [10] .

Superoxide Dismutase (SOD)

A significant (p < 0.05) increase in superoxide dismutase concentration was noted with Spirulina supplementation.

Birds supplemented with 1% Spirulina exhibited the highest SOD activity, which was significantly higher than that of control group and Antibiotic group. The T3 and T4 groups showed intermediate values that did not differ significantly from either the highest (T5) or the lower performing treatments. Abdel-Moneim et al. (2022) reported that SOD activity increased significantly with Spirulina inclusion, with both linear and quadratic components evident, demonstrating strong antioxidant enzyme stimulation [2] . Alaqil et al.

(2023) reported that supplementation with Spirulina increased antioxidant markers including elevated SOD levels while reducing oxidative stress metabolites [4] . Hassan et al. (2024) observed strong improvements with significantly increased SOD activity MDA levels [5] . The enhanced antioxidant status observed in the present study likely contributes to improved growth performance and feed efficiency, as reduced oxidative stress supports optimal cellular function, protein synthesis and energy metabolism.

Conclusion

Based on the observed results, it was concluded that Spirulina supplementation at higher inclusion levels, particularly 1%, exerted a greater synergistic influence on the evaluated parameters. This level was found to be the most effective in enhancing immunity, strengthening antioxidant defense mechanisms, thereby supporting its use as a natural alternative to antibiotic growth promoters in broiler diets.

Acknowledgement

AcknowledgementThe authors are thankful to Department of Poultry Science, CVSc, PV Narsimha Rao Telangana Veterinary University, R'nagar, Hyderabad, India for providing research facilities and techinical support for the conducting the experiment.

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