Indian Journal of Animal Research

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Dietary Spirulina Supplementation Enhanced Performance, Immune Response and Reduced Oxidative Stress in Broiler Chicken

M. Naik1, K. Sethy1,*, N. Panda1, S.K. Mishra1, V.T. Andhale1, D. Thakur1
1Department of Animal Nutrition, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.

ABSTRACT

Background: Spirulina have antimicrobial, antioxidant, anticancer, anti-inflammatory and immune-enhancing properties, which improved the performance of birds. It has also got metallo protective, radio protective, hypocholesterolemic and antiviral properties.

Methods: A 35-day study was conducted, taking one hundred sixty Vencobb broiler birds divided into four groups. Each group consists of four replicates with 10 birds each. The dietary treatments were: T0: basal diet; T0.5: basal diet + 0.5% Spirulina powder; T1.0: basal diet + 1.0% Spirulina powder and T1.5: basal diet + 1.5% Spirulina powder. Parameters under study included body weight gain, cumulative FCR, antioxidants, immunity status and intestinal E. coli, Salmonella, and Lactobacillus counts.

Result: Spirulina supplementation at 1.5% significantly (P<0.05) improved the body weight gain, FCR, antioxidant status, immunity, and intestinal Lactobacillus count in broiler chickens compared to other treatments. Supplementation of Spirulina at 0.5% and 1% showed better performance compared to un-supplemented control birds.

INTRODUCTION

Microorganisms like bacteria, fungi, microalgae, etc. play a great role in meeting the nutritional requirements of human beings, livestock, and poultry. Among all the microorganisms, algae are the chief photosynthetic producers. The most commonly used algae in animal and poultry feed is Spirulina platensis (Seyidoglu et al., 2020).

It is a unicellular filamentous cyanobacterium that generally occurs in a spiral shape (Holman et al., 2012).The excellent nutrient profile of Spirulina in terms of high carotenoid, iron, and phosphorus content in addition to protein is responsible for its potential growth-promoting effect (Solanki et al., 2023). Spirulina is also a non-toxic, nutritious feed for reproduction and immune enhancement in animals and poultry. It is also rich in all essential amino acids, vitamins, and gamma-linolenic acids (GLA), which are highly required for the health benefits of livestock (Howe et al., 2006).

Spirulina also produces different biologically active products that are responsible for anti-oxidant, anti-inflammatory, and anti-cancerous effects (Khanna et al., 2016). These bioactive products are phenolic compounds, like phycobillin proteins and phycocyanin C (Khadanga et al., 2023). Supplementation with Spirulina enhanced cell-mediated and humoral immunity in birds (Khadanga et al., 2023). It improves the mononuclear phagocytic potential in chickens for better resistance to various diseases (Seyidoglu et al., 2017). It also enhances the defense mechanism of the organism by increasing the phagocytic cell number, which ultimately increases the phagocytic activity and decreases the local infection without interfering with the natural defense system (Finamore et al., 2017). It also enhances feed conversion efficiency, average daily weight gain and carcass yield percentage in chickens (Kharde et al., 2012).

During recent research, a carbohydrate extracted from Spirulina platensis, known as “spirulin,” was found to popularly act as a prebiotic for the beneficial microbiota present inside the body of the organism (Sugiharto et al., 2018). It inhibits as well as promotes different microorganisms in the body due to the production of different acids and bioactive products. It inhibits several bacteria like Staphylococcus faecalis, S.epidermitis, Candida albicans, gram +ve bacteria like S. aureus, gram -ve bacteria E.coli and promotes the probiotic effect enhancing bacteria widely known as lactic acid bacteria which include L. lactis, L. casei, L. acdidophilus, L.bulgaricus (Finamore et al., 2017). It also improves the absorption of minerals, protects from diarrhea, and optimizes nutrient digestion processes (Gruzauskas et al., 2004). However, the supplementation of Spirulina and its effect on broiler chickens are relatively scanty and largely unknown in Indian context. Therefore; the present experiment was designed to assess the effect of Spirulina supplementation on the performance of Vencobb broiler chickens.

MATERIALS AND METHODS

Selection of birds and dietary treatments
 
The present study of 35 days duration was carried out as per the guidelines laid down by Institutional Animal Ethics Committee, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar during the month of March and April, 2024. 160-day-old Vencobb broiler chicks were randomly assigned to four dietary treatments. Each treatment had four replicas, each with 10 chicks. In the T0 group, birds were fed the regular basal diet. In T0.5, T1.0 and T1.5, birds were fed a basal diet similar to that of the control in addition to Spirulina powder at 0.5%, 1.0% and 1.50% of the basal diet, respectively. A deep litter system was used to rear the chicks. The experimental diets were made in accordance with BIS (2007). Before the experiment, the pen, feeders, and waterers were properly cleaned with water, followed by disinfection with Germex (Vetneeds Lab). Birds were given the RD (Lasota strain) vaccination on the seventh day, the IBD (intermediate strain) vaccine on the 14th day, and a booster dose of RD on the 21st day.
 
Preparation of spirulina powder
 
Spirulina was cultivated in the month of July, 2023 in a modified Zarrouk’s medium (NaCl 1.0 g/L; CaCl2.2H2O 0.04 g/L; KNO3 2.5 g/L; FeSO4.7H2O 0.01 g/L; EDTA (Na) 0.08 g/L; K2SO4 1.0 g/L; MgSO4.7H2O 0.2 g/L; NaHCO3 16.8 g/L and K2HPO4 0.5 g/L). Algae were incubated in an artificial pond at mean temperature of 30oC. Harvesting was performed after 12-14 days. It was dried at 40oC and the composition of Spirulina powder was measured by AOAC (1995).
 
Body weight gain and FCR
 
Individual body weights of the birds were measured using an electric pan balance at different weeks of the experiments. The initial body weight was subtracted from the final to get the BW gain. Feed intake was calculated at a weekly interval by subtracting the leftover feed from the total feed supplied during that week. FCR was calculated using a standard formula.
 
Estimation of antioxidant enzymes
 
Lipid peroxidation in RBC hemolyzate was determined by Placer et al., (1966), wherein the concentration of malonaldialdehyde (MDA) in nmol of MDA/mg hemoglobin was calculated using the extinction coefficient of 1.56 x 108/M/cm (Utely et al., 1967). Catalase (CAT) was assayed in erythrocytes by the method of Bergmeyer (1983). The superoxide dismutase (SOD) activity of RBC haemolyzate samples was measured using nitro blue tetrazolium as a substrate after suitable dilution according to Marklund and Marklund (1974) with certain modifications as suggested by Minami and Yoshikawa (1979).
 
Immunity study
 
The cellular immunity was assessed at the age of five weeks (Edelman et al., 1986) by injecting PHA-P intradermally in the foot web of two birds from each replicate (eight birds in each treatment). The humoral immunity was determined after the fifth week of the experiment as per the method of Khalifeh et al., (2009).
 
Estimation of bacterial load
 
Caecal samples collected from experimental birds were serially diluted up to a level of 10-6. Then, 0.1 ml of the dilution was pipetted out and inoculated in McConkey’s agar, Eosin-Methylene blue agar, and Lactobacillus MRS agar for the growth of Salmonella, E. coli, and Lactobacillus, respectively. The samples were applied to the agar surface using a sterilized glass spreader while simultaneously rotating the petri dish beneath. The agar containing petridish was incubated at 37oC for 24 h, and individual colonies were counted with a colony counter and quantified as:
 
 
 
 Statistical analysis
 
Statistical analysis was done using Software Package for Social Sciences (SPSS) Version 17.0 (2008), and one-way analysis of variance (generalized linear model, ANOVA) with comparison among means was made by Duncan’s multiple range test (Duncan, 1955) with a significance level of P≤0.05.

RESULTS AND DISCUSSION

Chemical composition of basal feed and spirulina
 
The broiler pre-starter, starter, and finisher rations had crude protein contents (%) of 22.50, 21.30 and 19.60, respectively (Table 1). The protein and energy needs were in line with those outlined in BIS (2007). Spirulina powder contained 46.50% CP and 7.40% EE. Similar to our result, Becker (2007) reported 46-63% crude protein and 4.0-9.0% ether extract in dry Spirulina. Ali (2017) observed higher protein content (61.8%) than our finding. This may be due to different growth mediums and climatic conditions during the cultivation.

Table 1: Composition of basal diet.


 
Body weight and feed conversion ratio
 
The day-old live body weight of experimental broiler chicks was almost alike, indicating the well-randomized distribution of chicks among the experimental treatments. It was seen that the body weight at the starter and finisher phases was significantly higher in all supplemented birds compared to the control. It was also observed that supplementation of Spirulina at 1.5% significantly improved body weight gain compared to 0.5% and 1.0% supplemented birds. Similarly, a better feed conversion ratio (FCR) was observed in supplemented birds compared to control during the starter and finisher phases (Table 2). This implies that supplementation of Spirulina1.5% in the ration of broilers had a significant (P<0.05) effect on body weight gain and FCR in birds.

​

Table 2: Body weight and FCR of experimental birds.



Similar to our results, significant improvement in body weight gain and FCR was observed in birds supplemented with varied levels of Spirulina (Jamil et al., 2015; Mirzaie et al., 2018; Park et al., 2018; Khan et al., 2021). El-Dayem  et al. (2021) and Ibrahim et al., (2018) also observed a significant (P<0.05) increase in body weight gain and a decreased feed conversion ratio when Spirulina was added at doses of 1 and 2 g/kg to the quail diet. Increased feed conversion efficiency in terms of lowered FCR is corroborated by the findings of Khan et al., (2021) and El-Dayem  et al.(2021). The increase in body weight in birds supplemented with Spirulina platensis may be due to improved feed utilization efficiency, vitamin and mineral absorption, and optimizing the nutrient digestion process (Gruzauskas et al., 2004). Spirulina have pre and probiotic effects, which enhances digestion, absorption and assimilation of various nutrients (El-Dayem  et al., 2021). 

Contrary to this, when Spirulina was provided to the broiler diet as a 10-20% replacement for soyabean meal, it showed a gradual decrease in growth rate (Toyomizu et al., 2001). The decreased body weight may be due to different strains, a high dose rate, and environmental conditions compared to the pre sent experiment.
 
Antioxidant status
 
Spirulina at different concentrations significantly (P<0.05) increased catalase and SOD levels and decreased (P<0.05) lipid peroxidation, as indicated by the fall in MDA levels in all the treatments compared to control birds (Table 3). Similarly, Moustafa et al., (2021) and Abd El-Hady et al., (2022) observed increased antioxidant enzyme activity and decreased malondialdehyde levels in chicken supplemented with Sprirulina. Omar et al., (2022) also observed that catalase and superoxide dismutase activity increased linearly and malondialdehyde (MDA) concentration decreased linearly by increasing the Spirulina level in chickens. The antioxidant activity of Spirulina is attributed to the presence of total phenols and flavonoids. Spirulina also contains C-phycocyanin, a potent antioxidant, as one of its major constituents (Mirzaie et al., 2018).

​

Table 3: Antioxidant status (U/mg haemoglobin) in experimental birds.



Immunity of birds
 
Spirulina supplementation enhanced the humoral and cellular immunity in broiler birds (Table 4). Similarly, Qureshi et al., (1996) and Lokapirnasari et al., (2016) observed significant improvement in immunity in birds supplemented with Spirulina. Spirulina enhances the function of macrophages and increases the potency of the mononuclear phagocyte system (Finamore et al., 2017). Active principles like β-glucan and phycocyanin pre sent in Spirulina enhanced the production and maturation of antibodies in birds (Seyidoglu et al., 2017). Supplementation of Spirulina platensis to K strain chicks at 0, 10, 100, 1,000, and 10,000 ppm improved the IgG and total anti-SRBC titers (P<0.05) over the controls. Supplementation of Spirulina significantly reduces the expression of cytokines like IL-1β, IL-6, and TNF-α ; C-phycocyanins and β-carotene pre sent in Spirulina block the activity of NF-κB and suppress TNF-α, leading to enhanced immunity in birds (El-Dayem et al., 2021).

​

Table 4: Immune status of experimental birds.


 
Intestinal microbial load
 
Log10 values of intestinal E. coli and Salmonella count were found to decrease significantly (P<0.05) in all the treated birds compared to the control (Fig 1). Earlier studies have suggested that Spirulinaand its extract are effective antimicrobial agents against bacteria, fungi, and viruses (Shanmugapriya et al., 2015; Fathi et al., 2018). The intestinal Lactobacillus count was found to be significantly (P<0.05) higher in all Spirulina-treated groups than in control birds.

Fig 1: Intestinal bacterial load of experimental birds at 35th day.



Similarly, Park et al., (2018) observed that supplementation with 1% Spirulina significantly increased the Lactobacillus count in birds. Fathi et al., (2018) observed that supplementation of Spirulina at 0.7 and 0.9 g/kg diet showed decreased microbial load and increased Lactobacillus count in the intestinal content. The non-digestible part of the cell wall of Spirulina acts as a source of prebiotics, so it helps in the growth of Lactobacillus.

CONCLUSION

Dietary supplementation of Spirulina at 1.5% resulted in maximum body weight gain, immunity and intestinal Lactobacillus count and reduced stress in birds. Addition of 0.5 % and 1.0 % Spirulina in the diet found to be beneficial for birds compared to control.

ACKNOWLEDGEMENT

The authors are thankful to the Vice Chancellor, Odisha University of Agriculture and Technology, Bhubaneswar for providing necessary funds and facilities to carry out this research.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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