Effect of dietary Spirulina (Arthrospira) platensis on the growth performance, antioxidant enzyme activity, nutrient digestibility, cecal microflora, excreta noxious gas emission, and breast meat quality of broiler chickens

Effect of dietary Spirulina (Arthrospira) platensis on the growth performance, antioxidant enzyme... ABSTRACT This study examined the effects of dietary Spirulina (Arthrospira) platensis supplementation on growth performance, antioxidant enzyme activity, nutrient digestibility, cecal microflora, excreta noxious gas emission, organ weight and breast meat quality in broiler chickens. In total, 800 Ross 308 male broiler chickens (1-d-old) were randomly divided into 5 dietary treatments with 10 replicate cages (16 birds/replicate) per treatment for 5 wk. The dietary treatments were a control basal diet without Spirulina or with 0.25, 0.5, 0.75, or 1.0% Spirulina. Body weight gain, feed conversion, and/or European production efficiency index improved linearly with supplementation of Spirulina during d 8 to 21, 22 to 35, and overall d 1 to 35 (P < 0.05). Dietary Spirulina supplementation caused a significant increase in the serum enzyme activity of superoxide dismutase and glutathione peroxidase (linear, P < 0.05). Apparent total tract digestibility of dry matter and nitrogen showed a linear increase in Spirulina supplementation (P < 0.05). Cecal Lactobacillus count linearly increased and excreta ammonia gas emission linearly decreased, as dietary Spirulina supplementation increased (P < 0.05). There were no significant effects on relative organ weight and breast meat quality of broilers fed with Spirulina diets; however, 7 d drip loss linearly decreased in treatment groups fed with Spirulina (P < 0.05). These results indicate that adding Spirulina to the diet of broilers can improve antioxidant enzyme activity, dry matter and nitrogen digestibility, cecal Lactobacillus population, excreta ammonia gas emission, and 7 d drip loss of breast meat. In addition, dietary inclusion of 1.0% Spirulina powder might provide a good alternative to improve broiler chicken production. INTRODUCTION Microalgae are attracting attention as the future clean energy and industrial material resources such as food, drug, cosmetics, and organic fertilizers because they can be mass-produced in a short time in various environments. In addition, Chlorella, Schizochytrium, and Spirulina are recognized as renewable substitutes for conventional protein sources (e.g., soybean meal, fish meal, rice bran) in aquaculture or animal feed because of their nutritional importance (Shields and Lupatsch, 2012). Spirulina (Arthrospira) platensis is a filamentous blue-green microalgae (cyanobacteria) generally regarded as a rich source of high quality protein, vitamins (particularly vitamin B12 and provitamin β-carotene), minerals, essential fatty acids, essential amino acids, pigments, and phenolic acids (Kulshreshtha et al., 2008; Bhavisha and Parula, 2010; Joventino et al., 2012). Many research studies have shown that Spirulina has antioxidant, immunomodulatory, anti-inflammatory, antiviral, and antimicrobial activity in various experimental animals (Rasool et al., 2009; Uyisenga et al., 2010; Langers et al., 2012; Abdel-Daim et al., 2013; Shokri et al., 2014). Recently, there has been a growing interest in its application in animals for its antioxidant activity, growth-promoting role, and immunomodulatory effects. These positive effects of Spirulina in the body may ultimately lead to improved animal productivity. For example, in feeding trials with livestock animals, Spirulina has been found to increase growth rate, nutrient utilization, disease resistance, egg quality, and carcass quality in poultry, pigs, and rabbit (Al-Batshan et al., 2001; Meineri et al., 2009; Sujatha and Narahari, 2011; Evans et al., 2015). However, present knowledge of broiler chicken response to dietary Spirulina supplementation is relatively unknown. The purpose of this study was to investigate the effect of Spirulina microalgae as a feed ingredient source in broiler chicken diets. Feeding experiments with broiler chickens were conducted to assess nutritional physiological properties, as well as to investigate effects on growth performance. MATERIALS AND METHODS Experimental protocols describing the management and care of animals were reviewed and approved by the Animal Care and Use Committee of Dankook University (Approval No. DK-1–1642), Republic of Korea. Animals and Housing A total of 800 male broiler chickens (1-d-old, Ross 308) were obtained from a commercial hatchery. Broiler chickens of similar body weight (41.5 ± 0.5 g) were randomly distributed into 5 groups (160 birds in 10 cages per treatment, 16 birds/cage). Broilers were housed in a temperature-controlled room with 3 floors of stainless steel battery cages (124 cm-width × 64 cm-length × 40 cm-height), which allowed free access to feed and water during the experimental period. They were kept in a room with controlled temperature and light regimen of 22L:2D for the entire experimental period. The environmental temperature was maintained at 33°C for the first week and then gradually reduced to 20°C by the fifth week. Relative humidity was gradually increased from 60% (d 1 to 21) to 70% (d 22 to 35). Diets Broilers were fed a corn/soybean-based basal diet for 35 d divided in 3 phases: Phase 1 (d 1 to 7), Phase 2 (d 8 to 21), and Phase 3 (d 22 to 35) (Table 1). The experimental diets, in mash form, were formulated to meet and exceed the nutrients requirements of NRC (1994) and Korean Feeding Standard for Poultry (2012). The dietary treatments were a control basal diet without Spirulina or with 0.25, 0.5, 0.75, or 1.0% Spirulina. A commercially available freeze-dried Spirulina powder was provided by a private company (NeoEnBiz Co., Bucheon, Republic of Korea) and supplemented to the basal diet, at the expense of soybean meal. Table 2 shows the nutrient composition of the freeze-dried Spirulina powder. Table 1. Ingredient composition of experimental diets (as-fed basis). Ingredients, % Phase 1 (d 1 to 7) Phase 2 (d 8 to 21) Phase 3 (d 22 to 35) Corn 35.22 39.82 37.56 Soybean meal 37.26 32.06 28.64 Wheat 15.00 15.00 20.00 Animal fat 4.15 5.07 6.61 Corn gluten meal 3.78 3.86 3.24 Monodicalcium phosphate 1.22 1.08 0.90 Limestone 1.80 1.64 1.61 Salt 0.36 0.36 0.63 Choline-chloride 0.14 0.13 0.12 L-Lysine 0.39 0.37 0.36 DL-Methionine 0.27 0.24 0.24 L-Threonine 0.09 0.07 0.07 Vit. Mix1 0.12 0.11 0.10 Min. Mix2 0.10 0.10 0.10 Phytase 0.10 0.10 0.10 Calculated values  ME, kcal/kg 3,000 3,100 3,200  CP, % 23.00 22.00 20.00  Dig. Lys, % 1.32 1.10 1.00  Dig. Met, % 0.52 0.50 0.38  Dig. Met + Cys, % 1.05 0.90 0.72  Dig. Thr, % 0.94 0.80 0.74  Dig. Iso, % 0.95 0.80 0.73  Dig. Val, % 1.09 0.90 0.82  Dig. Arg, % 1.45 1.25 1.10  Ca, % 1.00 1.00 0.90  Total P, % 0.80 0.80 0.75  Available P, % 0.45 0.45 0.35  Na, % 0.20 0.20 0.15 Analyzed values  CP, % 23.87 21.87 20.28  Lys, % 1.42 1.27 1.17  Met, % 0.64 0.58 0.56  Ca, % 1.05 0.92 0.85  P, % 0.74 0.76 0.72 Ingredients, % Phase 1 (d 1 to 7) Phase 2 (d 8 to 21) Phase 3 (d 22 to 35) Corn 35.22 39.82 37.56 Soybean meal 37.26 32.06 28.64 Wheat 15.00 15.00 20.00 Animal fat 4.15 5.07 6.61 Corn gluten meal 3.78 3.86 3.24 Monodicalcium phosphate 1.22 1.08 0.90 Limestone 1.80 1.64 1.61 Salt 0.36 0.36 0.63 Choline-chloride 0.14 0.13 0.12 L-Lysine 0.39 0.37 0.36 DL-Methionine 0.27 0.24 0.24 L-Threonine 0.09 0.07 0.07 Vit. Mix1 0.12 0.11 0.10 Min. Mix2 0.10 0.10 0.10 Phytase 0.10 0.10 0.10 Calculated values  ME, kcal/kg 3,000 3,100 3,200  CP, % 23.00 22.00 20.00  Dig. Lys, % 1.32 1.10 1.00  Dig. Met, % 0.52 0.50 0.38  Dig. Met + Cys, % 1.05 0.90 0.72  Dig. Thr, % 0.94 0.80 0.74  Dig. Iso, % 0.95 0.80 0.73  Dig. Val, % 1.09 0.90 0.82  Dig. Arg, % 1.45 1.25 1.10  Ca, % 1.00 1.00 0.90  Total P, % 0.80 0.80 0.75  Available P, % 0.45 0.45 0.35  Na, % 0.20 0.20 0.15 Analyzed values  CP, % 23.87 21.87 20.28  Lys, % 1.42 1.27 1.17  Met, % 0.64 0.58 0.56  Ca, % 1.05 0.92 0.85  P, % 0.74 0.76 0.72 1Provided per kg of complete diet: 11,025 IU of vitamin A; 1,103 IU of vitamin D3; 44 IU of vitamin E; 4.4 mg of vitamin K; 8.3 mg of riboflavin; 50 mg of niacin; 4 mg of thiamine; 29 mg of d-pantothenic; 166 mg of choline; 33 μg of vitamin B12. 2Provided per kg of complete diet: 12 mg of Cu (as CuSO4.5H2O); 85 mg of Zn (as ZnSO4); 8 mg of Mn (as MnO2); 0.28 mg of I (as KI); 0.15 mg of Se (as Na2SeO3.5H2O). View Large Table 1. Ingredient composition of experimental diets (as-fed basis). Ingredients, % Phase 1 (d 1 to 7) Phase 2 (d 8 to 21) Phase 3 (d 22 to 35) Corn 35.22 39.82 37.56 Soybean meal 37.26 32.06 28.64 Wheat 15.00 15.00 20.00 Animal fat 4.15 5.07 6.61 Corn gluten meal 3.78 3.86 3.24 Monodicalcium phosphate 1.22 1.08 0.90 Limestone 1.80 1.64 1.61 Salt 0.36 0.36 0.63 Choline-chloride 0.14 0.13 0.12 L-Lysine 0.39 0.37 0.36 DL-Methionine 0.27 0.24 0.24 L-Threonine 0.09 0.07 0.07 Vit. Mix1 0.12 0.11 0.10 Min. Mix2 0.10 0.10 0.10 Phytase 0.10 0.10 0.10 Calculated values  ME, kcal/kg 3,000 3,100 3,200  CP, % 23.00 22.00 20.00  Dig. Lys, % 1.32 1.10 1.00  Dig. Met, % 0.52 0.50 0.38  Dig. Met + Cys, % 1.05 0.90 0.72  Dig. Thr, % 0.94 0.80 0.74  Dig. Iso, % 0.95 0.80 0.73  Dig. Val, % 1.09 0.90 0.82  Dig. Arg, % 1.45 1.25 1.10  Ca, % 1.00 1.00 0.90  Total P, % 0.80 0.80 0.75  Available P, % 0.45 0.45 0.35  Na, % 0.20 0.20 0.15 Analyzed values  CP, % 23.87 21.87 20.28  Lys, % 1.42 1.27 1.17  Met, % 0.64 0.58 0.56  Ca, % 1.05 0.92 0.85  P, % 0.74 0.76 0.72 Ingredients, % Phase 1 (d 1 to 7) Phase 2 (d 8 to 21) Phase 3 (d 22 to 35) Corn 35.22 39.82 37.56 Soybean meal 37.26 32.06 28.64 Wheat 15.00 15.00 20.00 Animal fat 4.15 5.07 6.61 Corn gluten meal 3.78 3.86 3.24 Monodicalcium phosphate 1.22 1.08 0.90 Limestone 1.80 1.64 1.61 Salt 0.36 0.36 0.63 Choline-chloride 0.14 0.13 0.12 L-Lysine 0.39 0.37 0.36 DL-Methionine 0.27 0.24 0.24 L-Threonine 0.09 0.07 0.07 Vit. Mix1 0.12 0.11 0.10 Min. Mix2 0.10 0.10 0.10 Phytase 0.10 0.10 0.10 Calculated values  ME, kcal/kg 3,000 3,100 3,200  CP, % 23.00 22.00 20.00  Dig. Lys, % 1.32 1.10 1.00  Dig. Met, % 0.52 0.50 0.38  Dig. Met + Cys, % 1.05 0.90 0.72  Dig. Thr, % 0.94 0.80 0.74  Dig. Iso, % 0.95 0.80 0.73  Dig. Val, % 1.09 0.90 0.82  Dig. Arg, % 1.45 1.25 1.10  Ca, % 1.00 1.00 0.90  Total P, % 0.80 0.80 0.75  Available P, % 0.45 0.45 0.35  Na, % 0.20 0.20 0.15 Analyzed values  CP, % 23.87 21.87 20.28  Lys, % 1.42 1.27 1.17  Met, % 0.64 0.58 0.56  Ca, % 1.05 0.92 0.85  P, % 0.74 0.76 0.72 1Provided per kg of complete diet: 11,025 IU of vitamin A; 1,103 IU of vitamin D3; 44 IU of vitamin E; 4.4 mg of vitamin K; 8.3 mg of riboflavin; 50 mg of niacin; 4 mg of thiamine; 29 mg of d-pantothenic; 166 mg of choline; 33 μg of vitamin B12. 2Provided per kg of complete diet: 12 mg of Cu (as CuSO4.5H2O); 85 mg of Zn (as ZnSO4); 8 mg of Mn (as MnO2); 0.28 mg of I (as KI); 0.15 mg of Se (as Na2SeO3.5H2O). View Large Table 2. Nutrient composition of freeze-dried Spirulina powder at −50°C. Items % Moisture 5.4 Protein 56.7 Fiber 0.01 Ash 7.8 Amino acid  Aspartate 6.2  Threonine 3.2  Serine 3.3  Glutamate 8.6  Proline 2.4  Glycine 3.1  Alanine 4.8  Valine 3.3  Methionine 1.2  Isoleucine 2.6  Leucine 5.2  Tyrosine 2.4  Phenylalanine 2.7  Lysine 3.1  Histidine 0.9  Arginine 3.7 Fatty acid  Lauric acid (C12:0) 0.4  Myristic acid (C14:0) 0.7  Palmitic acid (C16:0) 15.5  Stearic acid (C18:0) 3.2  Arachidic acid (C20:0) 0.4  Behenic acid (C22:0) 0.4  Lignoceric acid (C24:0) 0.4  Palmitoleic acid (C16:1) 0.7  Oleic acid (C18:1) 32.5  Linoleic acid (C18:2n−6) 22.7  α-Linolenic acid (C18:3n−3) 21.4  Gadoleic acid (C20:1) 0.6 Items % Moisture 5.4 Protein 56.7 Fiber 0.01 Ash 7.8 Amino acid  Aspartate 6.2  Threonine 3.2  Serine 3.3  Glutamate 8.6  Proline 2.4  Glycine 3.1  Alanine 4.8  Valine 3.3  Methionine 1.2  Isoleucine 2.6  Leucine 5.2  Tyrosine 2.4  Phenylalanine 2.7  Lysine 3.1  Histidine 0.9  Arginine 3.7 Fatty acid  Lauric acid (C12:0) 0.4  Myristic acid (C14:0) 0.7  Palmitic acid (C16:0) 15.5  Stearic acid (C18:0) 3.2  Arachidic acid (C20:0) 0.4  Behenic acid (C22:0) 0.4  Lignoceric acid (C24:0) 0.4  Palmitoleic acid (C16:1) 0.7  Oleic acid (C18:1) 32.5  Linoleic acid (C18:2n−6) 22.7  α-Linolenic acid (C18:3n−3) 21.4  Gadoleic acid (C20:1) 0.6 View Large Table 2. Nutrient composition of freeze-dried Spirulina powder at −50°C. Items % Moisture 5.4 Protein 56.7 Fiber 0.01 Ash 7.8 Amino acid  Aspartate 6.2  Threonine 3.2  Serine 3.3  Glutamate 8.6  Proline 2.4  Glycine 3.1  Alanine 4.8  Valine 3.3  Methionine 1.2  Isoleucine 2.6  Leucine 5.2  Tyrosine 2.4  Phenylalanine 2.7  Lysine 3.1  Histidine 0.9  Arginine 3.7 Fatty acid  Lauric acid (C12:0) 0.4  Myristic acid (C14:0) 0.7  Palmitic acid (C16:0) 15.5  Stearic acid (C18:0) 3.2  Arachidic acid (C20:0) 0.4  Behenic acid (C22:0) 0.4  Lignoceric acid (C24:0) 0.4  Palmitoleic acid (C16:1) 0.7  Oleic acid (C18:1) 32.5  Linoleic acid (C18:2n−6) 22.7  α-Linolenic acid (C18:3n−3) 21.4  Gadoleic acid (C20:1) 0.6 Items % Moisture 5.4 Protein 56.7 Fiber 0.01 Ash 7.8 Amino acid  Aspartate 6.2  Threonine 3.2  Serine 3.3  Glutamate 8.6  Proline 2.4  Glycine 3.1  Alanine 4.8  Valine 3.3  Methionine 1.2  Isoleucine 2.6  Leucine 5.2  Tyrosine 2.4  Phenylalanine 2.7  Lysine 3.1  Histidine 0.9  Arginine 3.7 Fatty acid  Lauric acid (C12:0) 0.4  Myristic acid (C14:0) 0.7  Palmitic acid (C16:0) 15.5  Stearic acid (C18:0) 3.2  Arachidic acid (C20:0) 0.4  Behenic acid (C22:0) 0.4  Lignoceric acid (C24:0) 0.4  Palmitoleic acid (C16:1) 0.7  Oleic acid (C18:1) 32.5  Linoleic acid (C18:2n−6) 22.7  α-Linolenic acid (C18:3n−3) 21.4  Gadoleic acid (C20:1) 0.6 View Large Growth Performance Body weight (BW) and feed intake (FI) per cage were recorded on d 7, 21, and 35, and the feed conversion ratio (FCR) was calculated based on feed intake divided by body weight gain (BWG). Mortality was recorded daily, and percentage mortality was calculated throughout the study. The European production efficiency index (EPEI) was calculated with following formula. EPEI = (BW/d × survival rate/FCR × 10). Antioxidant Enzyme Activity Analysis At the end of the experiment (35 d), blood samples were collected from the left wing vein into K3EDTA vacuum tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ), and stored at 4°C. For serum analysis, approximately 3 mL of blood samples were centrifuged at 4,000 × g for 15 min at 4°C, after which the serum was separated. Antioxidant enzyme activities, superoxide dismutase (SOD), and glutathione peroxidase (GPx) in serum were measured using a commercial kit from Cayman Chemical Company (Cayman Chemical Co., Ann Arbor, MI, USA), according to the manufacturer's instructions. Apparent Total Tract Digestibility To determine the apparent total tract digestibility, 0.2% chromic oxide was added to the experimental diets 4 d prior to the collection period. Excreta were collected daily for the last 3 d of the experiment, and placed into a 60°C oven for 72 h. After drying, excreta were pulverized to pass through a 1-mm screen, and dry matter and nitrogen in diets and excreta were analyzed (methods 934.01 and 968.06; AOAC, 2000). Chromium concentration was determined by atomic absorption spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan). The equation for calculating digestibility was as follows: digestibility (%) = (1 – ((Nf × Cd)/(Nd × Cf))) × 100, where Nf = nutrient concentration in feces (% DM), Nd = nutrient concentration in diet (% DM), Cf = chromium concentration in feces (% DM), and Cd = chromium concentration in diet (% DM). Cecal Microflora Population One gram of cecal sample was blended with 9 mL of sterile peptone water and mixed for 1 min on a vortex stirrer. Viable counts of bacteria in the cecal samples were conducted by plating serial 10-fold dilutions (10−1 to 10−8) onto Lactobacilli MRS agar (Difco Laboratories, Detroit, MI, USA) plates and MacConkey agar (Difco Laboratories, Detroit, MI, USA) plates to isolate Lactobacillus spp. and coliform bacteria, respectively. The lactobacilli agar plates were then incubated for 48 h at 37°C under anaerobic conditions. The MacConkey agar plates were incubated for 24 h at 37°C under aerobic conditions. After the incubation periods, colonies of the respective bacteria were counted and expressed as the logarithm of colony-forming units per gram (log10 CFU/g). Excreta Noxious Gas Emission During the last 3 d of the experiment, fresh excreta samples were collected from each replication for analyzing ammonia, hydrogen sulfide, and total mercaptan. The excreta samples were kept in 3 L sealed plastic containers for 5 d at room temperature (24°C). After the fermentation period, a Gastec (model GV-100) gas sampling pump was utilized for gas detection (Gastec Corp., Tokyo, Japan). Concentrations of ammonia, hydrogen sulfide, and total mercaptan were measured within the scope of 5.0 to 100.0 (No. 3La, detector tube; Gastec Corp.), 2.0 to 20.0 (No. 4LK, detector tube; Gastec Corp.), and 0.5 to 120.0 (No.70 and 70-L, detector tubes; Gastec Corp.) ppm. The adhesive plaster was punctured, and 100 mL of headspace air was sampled at approximately 3 cm above the excreta. Breast Meat Quality and Relative Organ Weight Color values of breast meat were measured in 3 replicates using a Minolta colorimeter (CR-300, Tokyo, Japan) calibrated with a standard white plate and recorded as L*, a*, and b* values for lightness, redness, and yellowness, respectively. The pH values of raw breast meat were measured using a pH meter (NWK Binar pH, K-21, Landsberg, Germany) after blending 10 g of finely homogenized sample with 90 mL of double-distilled water. To estimate the cooking loss, raw meat samples were packed into Cryovac Cook-In Bags after weighing, and cooked in a water bath at 100°C for 30 min. Samples were cooled at room temperature for 1 h and reweighed. Cooking loss was calculated as the weight difference between the initial raw and final cooked samples. Water-holding capacity (WHC) was determined following the method of Kristensen and Purslow et al. (2001). Five grams of meat sample was heated to 70°C in a water bath for 30 min. Samples were then cooled with ice and subsequently centrifuged at 4°C at 1,000 × g for 10 min. WHC (%) was calculated as the ratio of weight loss of the sample during centrifugation, to that of the original liquid. Drip loss (%) was measured for 3 cm × 3 cm cuts of breast meat, which were weighed, hung in a zipper bag, and stored at 4°C. After storage, moisture on the surface of the meat slices was carefully removed and weighed at d 1, 3, 5, and 7 after the sample was taken. The initial and final weight of each sample was used to calculate drip loss. The liver, spleen, bursa of Fabricius, breast meat, abdominal fat, and gizzard were removed and weighed. Organ weights, breast meat, and abdominal fat were expressed as a percentage of live BW. Statistical Analysis All data were statistically analyzed using the GLM procedure in SAS program (SAS Institute Inc., Cary, NC). Polynomial contrasts were used to determine linear, quadratic, cubic, and quartic effects of increasing Spirulina levels on all measurements. Cage was used as an experimental unit for growth performance, nutrient digestibility, and excreta noxious gas. The individual bird was used as the experimental unit for blood oxidant enzyme, cecal microflora, and meat quality measurements. Alpha was set at 0.05. RESULTS Growth Performance Dietary Spirulina supplementation linearly increased for BWG during d 8 to 21, 22 to 35, and overall d 1 to 35, as the inclusion rate increased from 0 to 1.0% (P < 0.05) (Table 3). Increasing dietary supplementation of Spirulina had a positive linear effect on the FCR during d 8 to 21 and overall (P < 0.05). The EPEI was linearly increased associated with the inclusion of graded levels of Spirulina in the diets. No treatment effects were observed on FI and mortality throughout all the phases of feeding. Table 3. The effect of dietary Spirulina supplementation on growth performance in broilers.1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM6 Linear Quadratic Cubic Quartic d 1 to 7  BWG2, g 80 82 80 83 84 1.30 0.0736 0.4412 0.5805 0.2245  FI3, g 105 105 104 104 103 1.66 0.3858 0.9489 0.8201 0.6992  FCR4 1.309 1.285 1.292 1.254 1.232 0.028 0.0426 0.6853 0.8665 0.5565 d 8 to 21  BWG, g 637 645 646 658 665 8.10 0.0117 0.7731 0.9536 0.6035  FI, g 997 999 1,003 1,013 994 9.29 0.8367 0.3088 0.2805 0.6226  FCR 1.568 1.549 1.556 1.539 1.496 0.020 0.0199 0.3344 0.4054 0.7668 d 22 to 35  BWG, g 1,011 1,033 1,035 1,046 1,052 10.70 0.0077 0.5776 0.6815 0.6497  FI, g 1,753 1,784 1,773 1,789 1,792 14.31 0.0759 0.6158 0.5242 0.3930  FCR 1.735 1.729 1.715 1.711 1.706 0.019 0.2216 0.8641 0.9064 0.8453 Overall  BWG, g 1,729 1,760 1,762 1,787 1,801 14.58 0.0003 0.8313 0.6945 0.4344  FI, g 2,856 2,888 2,880 2,906 2,889 18.37 0.1507 0.3573 0.9714 0.3424  FCR 1.653 1.642 1.636 1.626 1.605 0.012 0.0044 0.5902 0.6570 0.9959  Mortality 5.2 5.3 5.0 4.4 4.2 0.59 0.7974 0.7811 0.6878 0.5317  EPEI5 284.2 291.4 292.6 301.7 308.0 4.08 0.0004 0.6911 0.7997 0.4758 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM6 Linear Quadratic Cubic Quartic d 1 to 7  BWG2, g 80 82 80 83 84 1.30 0.0736 0.4412 0.5805 0.2245  FI3, g 105 105 104 104 103 1.66 0.3858 0.9489 0.8201 0.6992  FCR4 1.309 1.285 1.292 1.254 1.232 0.028 0.0426 0.6853 0.8665 0.5565 d 8 to 21  BWG, g 637 645 646 658 665 8.10 0.0117 0.7731 0.9536 0.6035  FI, g 997 999 1,003 1,013 994 9.29 0.8367 0.3088 0.2805 0.6226  FCR 1.568 1.549 1.556 1.539 1.496 0.020 0.0199 0.3344 0.4054 0.7668 d 22 to 35  BWG, g 1,011 1,033 1,035 1,046 1,052 10.70 0.0077 0.5776 0.6815 0.6497  FI, g 1,753 1,784 1,773 1,789 1,792 14.31 0.0759 0.6158 0.5242 0.3930  FCR 1.735 1.729 1.715 1.711 1.706 0.019 0.2216 0.8641 0.9064 0.8453 Overall  BWG, g 1,729 1,760 1,762 1,787 1,801 14.58 0.0003 0.8313 0.6945 0.4344  FI, g 2,856 2,888 2,880 2,906 2,889 18.37 0.1507 0.3573 0.9714 0.3424  FCR 1.653 1.642 1.636 1.626 1.605 0.012 0.0044 0.5902 0.6570 0.9959  Mortality 5.2 5.3 5.0 4.4 4.2 0.59 0.7974 0.7811 0.6878 0.5317  EPEI5 284.2 291.4 292.6 301.7 308.0 4.08 0.0004 0.6911 0.7997 0.4758 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Body weight gain. 3Feed intake. 4Feed conversion ratio. 5European production efficiency index. 6Standard error of means. View Large Table 3. The effect of dietary Spirulina supplementation on growth performance in broilers.1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM6 Linear Quadratic Cubic Quartic d 1 to 7  BWG2, g 80 82 80 83 84 1.30 0.0736 0.4412 0.5805 0.2245  FI3, g 105 105 104 104 103 1.66 0.3858 0.9489 0.8201 0.6992  FCR4 1.309 1.285 1.292 1.254 1.232 0.028 0.0426 0.6853 0.8665 0.5565 d 8 to 21  BWG, g 637 645 646 658 665 8.10 0.0117 0.7731 0.9536 0.6035  FI, g 997 999 1,003 1,013 994 9.29 0.8367 0.3088 0.2805 0.6226  FCR 1.568 1.549 1.556 1.539 1.496 0.020 0.0199 0.3344 0.4054 0.7668 d 22 to 35  BWG, g 1,011 1,033 1,035 1,046 1,052 10.70 0.0077 0.5776 0.6815 0.6497  FI, g 1,753 1,784 1,773 1,789 1,792 14.31 0.0759 0.6158 0.5242 0.3930  FCR 1.735 1.729 1.715 1.711 1.706 0.019 0.2216 0.8641 0.9064 0.8453 Overall  BWG, g 1,729 1,760 1,762 1,787 1,801 14.58 0.0003 0.8313 0.6945 0.4344  FI, g 2,856 2,888 2,880 2,906 2,889 18.37 0.1507 0.3573 0.9714 0.3424  FCR 1.653 1.642 1.636 1.626 1.605 0.012 0.0044 0.5902 0.6570 0.9959  Mortality 5.2 5.3 5.0 4.4 4.2 0.59 0.7974 0.7811 0.6878 0.5317  EPEI5 284.2 291.4 292.6 301.7 308.0 4.08 0.0004 0.6911 0.7997 0.4758 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM6 Linear Quadratic Cubic Quartic d 1 to 7  BWG2, g 80 82 80 83 84 1.30 0.0736 0.4412 0.5805 0.2245  FI3, g 105 105 104 104 103 1.66 0.3858 0.9489 0.8201 0.6992  FCR4 1.309 1.285 1.292 1.254 1.232 0.028 0.0426 0.6853 0.8665 0.5565 d 8 to 21  BWG, g 637 645 646 658 665 8.10 0.0117 0.7731 0.9536 0.6035  FI, g 997 999 1,003 1,013 994 9.29 0.8367 0.3088 0.2805 0.6226  FCR 1.568 1.549 1.556 1.539 1.496 0.020 0.0199 0.3344 0.4054 0.7668 d 22 to 35  BWG, g 1,011 1,033 1,035 1,046 1,052 10.70 0.0077 0.5776 0.6815 0.6497  FI, g 1,753 1,784 1,773 1,789 1,792 14.31 0.0759 0.6158 0.5242 0.3930  FCR 1.735 1.729 1.715 1.711 1.706 0.019 0.2216 0.8641 0.9064 0.8453 Overall  BWG, g 1,729 1,760 1,762 1,787 1,801 14.58 0.0003 0.8313 0.6945 0.4344  FI, g 2,856 2,888 2,880 2,906 2,889 18.37 0.1507 0.3573 0.9714 0.3424  FCR 1.653 1.642 1.636 1.626 1.605 0.012 0.0044 0.5902 0.6570 0.9959  Mortality 5.2 5.3 5.0 4.4 4.2 0.59 0.7974 0.7811 0.6878 0.5317  EPEI5 284.2 291.4 292.6 301.7 308.0 4.08 0.0004 0.6911 0.7997 0.4758 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Body weight gain. 3Feed intake. 4Feed conversion ratio. 5European production efficiency index. 6Standard error of means. View Large Antioxidant Enzyme Activity Antioxidant enzyme activity of Spirulina was evaluated by analyzing serum SOD and GPx, and a linear increase in these enzymes was observed with increasing dietary levels of Spirulina (P < 0.0016 and P < 0.0001) (Table 4). Table 4. The effect of dietary Spirulina supplementation on blood SOD and GPx in broilers (d 35).1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM4 Linear Quadratic Cubic Quartic SOD2, U/mL 4.4 5.5 5.8 5.9 6.2 0.31 0.0016 0.1332 0.3568 0.9541 GPx3, mU/mL 38.6 44.7 43.2 46.0 45.6 0.76 <0.0001 0.0096 0.0794 0.0109 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM4 Linear Quadratic Cubic Quartic SOD2, U/mL 4.4 5.5 5.8 5.9 6.2 0.31 0.0016 0.1332 0.3568 0.9541 GPx3, mU/mL 38.6 44.7 43.2 46.0 45.6 0.76 <0.0001 0.0096 0.0794 0.0109 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Superoxide dismutase. 3Glutathione peroxidase. 4Standard error of means. View Large Table 4. The effect of dietary Spirulina supplementation on blood SOD and GPx in broilers (d 35).1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM4 Linear Quadratic Cubic Quartic SOD2, U/mL 4.4 5.5 5.8 5.9 6.2 0.31 0.0016 0.1332 0.3568 0.9541 GPx3, mU/mL 38.6 44.7 43.2 46.0 45.6 0.76 <0.0001 0.0096 0.0794 0.0109 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM4 Linear Quadratic Cubic Quartic SOD2, U/mL 4.4 5.5 5.8 5.9 6.2 0.31 0.0016 0.1332 0.3568 0.9541 GPx3, mU/mL 38.6 44.7 43.2 46.0 45.6 0.76 <0.0001 0.0096 0.0794 0.0109 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Superoxide dismutase. 3Glutathione peroxidase. 4Standard error of means. View Large Apparent Total Tract Digestibility The apparent total tract digestibility of dry matter and nitrogen linearly increased in broiler chickens fed diets supplemented with 0 to 1.0% Spirulina (P < 0.05) (Table 5). Table 5. The effect of dietary Spirulina supplementation on apparent total tract nutrient digestibility in broilers.1 Spirulina, % P-value Items, % 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Dry matter 67.9 68.5 69.7 70.1 71.1 0.68 0.0325 0.7084 0.7160 0.9944 Nitrogen 66.1 66.6 67.0 67.9 68.7 0.78 0.0157 0.6724 0.9796 0.8838 Spirulina, % P-value Items, % 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Dry matter 67.9 68.5 69.7 70.1 71.1 0.68 0.0325 0.7084 0.7160 0.9944 Nitrogen 66.1 66.6 67.0 67.9 68.7 0.78 0.0157 0.6724 0.9796 0.8838 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Standard error of means. View Large Table 5. The effect of dietary Spirulina supplementation on apparent total tract nutrient digestibility in broilers.1 Spirulina, % P-value Items, % 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Dry matter 67.9 68.5 69.7 70.1 71.1 0.68 0.0325 0.7084 0.7160 0.9944 Nitrogen 66.1 66.6 67.0 67.9 68.7 0.78 0.0157 0.6724 0.9796 0.8838 Spirulina, % P-value Items, % 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Dry matter 67.9 68.5 69.7 70.1 71.1 0.68 0.0325 0.7084 0.7160 0.9944 Nitrogen 66.1 66.6 67.0 67.9 68.7 0.78 0.0157 0.6724 0.9796 0.8838 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Standard error of means. View Large Cecal Microbial Count There was no significant difference in coliform bacteria counts of broiler chickens fed with different levels of Spirulina. However, Lactobacillus counts were significantly increased linearly as dietary Spirulina supplementation increased (P < 0.05) (Table 6). Table 6. The effect of dietary Spirulina supplementation on cecal microflora in broilers (d 35).1 Spirulina, % P-value Items, log10 cfu/g 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Lactobacillus 7.39 7.45 7.50 7.63 7.78 0.10 0.0092 0.4988 0.9625 0.8822 Coliforms 5.05 5.03 5.02 5.02 4.99 0.03 0.1179 0.9852 0.7172 0.8297 Spirulina, % P-value Items, log10 cfu/g 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Lactobacillus 7.39 7.45 7.50 7.63 7.78 0.10 0.0092 0.4988 0.9625 0.8822 Coliforms 5.05 5.03 5.02 5.02 4.99 0.03 0.1179 0.9852 0.7172 0.8297 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Standard error of means. View Large Table 6. The effect of dietary Spirulina supplementation on cecal microflora in broilers (d 35).1 Spirulina, % P-value Items, log10 cfu/g 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Lactobacillus 7.39 7.45 7.50 7.63 7.78 0.10 0.0092 0.4988 0.9625 0.8822 Coliforms 5.05 5.03 5.02 5.02 4.99 0.03 0.1179 0.9852 0.7172 0.8297 Spirulina, % P-value Items, log10 cfu/g 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Lactobacillus 7.39 7.45 7.50 7.63 7.78 0.10 0.0092 0.4988 0.9625 0.8822 Coliforms 5.05 5.03 5.02 5.02 4.99 0.03 0.1179 0.9852 0.7172 0.8297 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Standard error of means. View Large Excreta Noxious Gas Emissions Excreta ammonia emissions decreased as dietary Spirulina supplementation increased (linear, P < 0.05) (Table 7). However, Spirulina supplementation did not affect total mercaptan or hydrogen sulfide emissions. Table 7. The effect of dietary Spirulina supplementation on excreta gas emission in broilers.1 Spirulina, % P-value Items, ppm 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Ammonia 45.1 35.9 34.1 31.0 31.2 0.26 0.0050 0.3213 0.6759 0.3082 Hydrogen sulfide 2.8 2.0 2.1 2.0 2.2 0.16 0.8497 0.4291 0.8869 0.5810 Mercaptan 3.3 2.5 2.1 2.4 2.8 0.77 0.9200 0.9729 0.9200 0.4987 Spirulina, % P-value Items, ppm 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Ammonia 45.1 35.9 34.1 31.0 31.2 0.26 0.0050 0.3213 0.6759 0.3082 Hydrogen sulfide 2.8 2.0 2.1 2.0 2.2 0.16 0.8497 0.4291 0.8869 0.5810 Mercaptan 3.3 2.5 2.1 2.4 2.8 0.77 0.9200 0.9729 0.9200 0.4987 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Standard error of means. View Large Table 7. The effect of dietary Spirulina supplementation on excreta gas emission in broilers.1 Spirulina, % P-value Items, ppm 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Ammonia 45.1 35.9 34.1 31.0 31.2 0.26 0.0050 0.3213 0.6759 0.3082 Hydrogen sulfide 2.8 2.0 2.1 2.0 2.2 0.16 0.8497 0.4291 0.8869 0.5810 Mercaptan 3.3 2.5 2.1 2.4 2.8 0.77 0.9200 0.9729 0.9200 0.4987 Spirulina, % P-value Items, ppm 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Ammonia 45.1 35.9 34.1 31.0 31.2 0.26 0.0050 0.3213 0.6759 0.3082 Hydrogen sulfide 2.8 2.0 2.1 2.0 2.2 0.16 0.8497 0.4291 0.8869 0.5810 Mercaptan 3.3 2.5 2.1 2.4 2.8 0.77 0.9200 0.9729 0.9200 0.4987 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Standard error of means. View Large Meat Quality and Organ Weight There were no significant differences in pH, color (L*, a*, b*), cooking loss, or WHC of breast meat among the 5 treatment groups (P > 0.05) (Table 8). However, drip loss at 7 d post slaughter was significantly different among the 5 groups. Birds fed with Spirulina showed significantly lower drip loss as dietary levels of Spirulina increased (linear, P < 0.05). Relative weights of most organs (liver, spleen, gizzard, and bursa of Fabricius), breast meat, and abdominal fat were not significantly influenced by dietary supplementation of Spirulina. Table 8. The effect of dietary Spirulina supplementation on meat quality and relative organ weight in broilers (d 35).1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM3 Linear Quadratic Cubic Quartic pH value 5.74 5.89 5.85 6.15 6.15 0.17 0.0708 0.9484 0.8500 0.4222 Breast muscle color  Lightness (L*) 41.60 45.49 45.14 43.18 46.47 1.34 0.1056 0.5878 0.0955 0.7116  Redness (a*) 10.44 10.07 10.80 11.07 10.29 0.49 0.6645 0.5033 0.1920 0.8133  Yellowness (b*) 9.33 9.64 9.93 8.46 9.86 0.77 0.9631 0.8878 0.2597 0.3436 Cooking loss 30.11 29.97 29.61 28.69 28.54 1.27 0.4882 0.9540 0.9484 0.9145 WHC2, % 40.05 39.53 39.16 38.39 37.61 2.39 0.3575 0.6348 0.9257 0.9452 Drip loss, %  d 1 5.73 5.59 5.54 5.51 5.36 0.72 0.7671 0.9667 0.8441 0.9246  d 3 9.19 8.90 8.79 8.68 8.65 0.26 0.1472 0.6068 0.9082 0.9296  d 5 11.34 10.92 10.86 10.82 10.76 0.28 0.1814 0.4833 0.6633 0.9004  d 7 13.34 13.05 12.93 12.88 12.82 0.09 0.0009 0.1174 0.4892 0.9760 Relative organ weight, %  Breast muscle 18.55 19.10 19.13 19.61 19.73 0.87 0.3156 0.9020 0.9529 0.8111  Liver 2.86 2.80 3.07 2.88 2.77 1.34 0.8885 0.4932 0.7157 0.4625  Bursa of Fabricius 0.17 0.14 0.13 0.15 0.15 0.77 0.5623 0.1967 0.4832 0.5627  Abdominal fat 3.29 2.91 2.85 2.81 2.70 0.34 0.2624 0.6639 0.7225 0.9389  Spleen 0.13 0.11 0.10 0.11 0.11 0.01 0.3614 0.1943 0.6437 0.8215  Gizzard 1.30 1.18 1.14 1.16 1.13 0.07 0.1420 0.3963 0.5769 0.8689 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM3 Linear Quadratic Cubic Quartic pH value 5.74 5.89 5.85 6.15 6.15 0.17 0.0708 0.9484 0.8500 0.4222 Breast muscle color  Lightness (L*) 41.60 45.49 45.14 43.18 46.47 1.34 0.1056 0.5878 0.0955 0.7116  Redness (a*) 10.44 10.07 10.80 11.07 10.29 0.49 0.6645 0.5033 0.1920 0.8133  Yellowness (b*) 9.33 9.64 9.93 8.46 9.86 0.77 0.9631 0.8878 0.2597 0.3436 Cooking loss 30.11 29.97 29.61 28.69 28.54 1.27 0.4882 0.9540 0.9484 0.9145 WHC2, % 40.05 39.53 39.16 38.39 37.61 2.39 0.3575 0.6348 0.9257 0.9452 Drip loss, %  d 1 5.73 5.59 5.54 5.51 5.36 0.72 0.7671 0.9667 0.8441 0.9246  d 3 9.19 8.90 8.79 8.68 8.65 0.26 0.1472 0.6068 0.9082 0.9296  d 5 11.34 10.92 10.86 10.82 10.76 0.28 0.1814 0.4833 0.6633 0.9004  d 7 13.34 13.05 12.93 12.88 12.82 0.09 0.0009 0.1174 0.4892 0.9760 Relative organ weight, %  Breast muscle 18.55 19.10 19.13 19.61 19.73 0.87 0.3156 0.9020 0.9529 0.8111  Liver 2.86 2.80 3.07 2.88 2.77 1.34 0.8885 0.4932 0.7157 0.4625  Bursa of Fabricius 0.17 0.14 0.13 0.15 0.15 0.77 0.5623 0.1967 0.4832 0.5627  Abdominal fat 3.29 2.91 2.85 2.81 2.70 0.34 0.2624 0.6639 0.7225 0.9389  Spleen 0.13 0.11 0.10 0.11 0.11 0.01 0.3614 0.1943 0.6437 0.8215  Gizzard 1.30 1.18 1.14 1.16 1.13 0.07 0.1420 0.3963 0.5769 0.8689 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Water holding capacity. 3Standard error of means. View Large Table 8. The effect of dietary Spirulina supplementation on meat quality and relative organ weight in broilers (d 35).1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM3 Linear Quadratic Cubic Quartic pH value 5.74 5.89 5.85 6.15 6.15 0.17 0.0708 0.9484 0.8500 0.4222 Breast muscle color  Lightness (L*) 41.60 45.49 45.14 43.18 46.47 1.34 0.1056 0.5878 0.0955 0.7116  Redness (a*) 10.44 10.07 10.80 11.07 10.29 0.49 0.6645 0.5033 0.1920 0.8133  Yellowness (b*) 9.33 9.64 9.93 8.46 9.86 0.77 0.9631 0.8878 0.2597 0.3436 Cooking loss 30.11 29.97 29.61 28.69 28.54 1.27 0.4882 0.9540 0.9484 0.9145 WHC2, % 40.05 39.53 39.16 38.39 37.61 2.39 0.3575 0.6348 0.9257 0.9452 Drip loss, %  d 1 5.73 5.59 5.54 5.51 5.36 0.72 0.7671 0.9667 0.8441 0.9246  d 3 9.19 8.90 8.79 8.68 8.65 0.26 0.1472 0.6068 0.9082 0.9296  d 5 11.34 10.92 10.86 10.82 10.76 0.28 0.1814 0.4833 0.6633 0.9004  d 7 13.34 13.05 12.93 12.88 12.82 0.09 0.0009 0.1174 0.4892 0.9760 Relative organ weight, %  Breast muscle 18.55 19.10 19.13 19.61 19.73 0.87 0.3156 0.9020 0.9529 0.8111  Liver 2.86 2.80 3.07 2.88 2.77 1.34 0.8885 0.4932 0.7157 0.4625  Bursa of Fabricius 0.17 0.14 0.13 0.15 0.15 0.77 0.5623 0.1967 0.4832 0.5627  Abdominal fat 3.29 2.91 2.85 2.81 2.70 0.34 0.2624 0.6639 0.7225 0.9389  Spleen 0.13 0.11 0.10 0.11 0.11 0.01 0.3614 0.1943 0.6437 0.8215  Gizzard 1.30 1.18 1.14 1.16 1.13 0.07 0.1420 0.3963 0.5769 0.8689 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM3 Linear Quadratic Cubic Quartic pH value 5.74 5.89 5.85 6.15 6.15 0.17 0.0708 0.9484 0.8500 0.4222 Breast muscle color  Lightness (L*) 41.60 45.49 45.14 43.18 46.47 1.34 0.1056 0.5878 0.0955 0.7116  Redness (a*) 10.44 10.07 10.80 11.07 10.29 0.49 0.6645 0.5033 0.1920 0.8133  Yellowness (b*) 9.33 9.64 9.93 8.46 9.86 0.77 0.9631 0.8878 0.2597 0.3436 Cooking loss 30.11 29.97 29.61 28.69 28.54 1.27 0.4882 0.9540 0.9484 0.9145 WHC2, % 40.05 39.53 39.16 38.39 37.61 2.39 0.3575 0.6348 0.9257 0.9452 Drip loss, %  d 1 5.73 5.59 5.54 5.51 5.36 0.72 0.7671 0.9667 0.8441 0.9246  d 3 9.19 8.90 8.79 8.68 8.65 0.26 0.1472 0.6068 0.9082 0.9296  d 5 11.34 10.92 10.86 10.82 10.76 0.28 0.1814 0.4833 0.6633 0.9004  d 7 13.34 13.05 12.93 12.88 12.82 0.09 0.0009 0.1174 0.4892 0.9760 Relative organ weight, %  Breast muscle 18.55 19.10 19.13 19.61 19.73 0.87 0.3156 0.9020 0.9529 0.8111  Liver 2.86 2.80 3.07 2.88 2.77 1.34 0.8885 0.4932 0.7157 0.4625  Bursa of Fabricius 0.17 0.14 0.13 0.15 0.15 0.77 0.5623 0.1967 0.4832 0.5627  Abdominal fat 3.29 2.91 2.85 2.81 2.70 0.34 0.2624 0.6639 0.7225 0.9389  Spleen 0.13 0.11 0.10 0.11 0.11 0.01 0.3614 0.1943 0.6437 0.8215  Gizzard 1.30 1.18 1.14 1.16 1.13 0.07 0.1420 0.3963 0.5769 0.8689 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Water holding capacity. 3Standard error of means. View Large DISCUSSION This study found that broiler chickens fed diets supplemented with Spirulina increased growth performance. The mechanism of action of Spirulina has not been clearly established, but previous studies have reported that dietary supplementation with Spirulina has positive effects on growth performance in poultry. Saxena et al. (1983) reported that White Leghorn chicks fed experimental diets containing 111 g/kg and 166 g/kg Spirulina had greater weight gains at 6 wks when Spirulina replaced groundnut cake. Venkataraman et al. (1994) reported that supplementation of 140 and 170 g/kg Spirulina, with no additional vitamins/minerals, could replace groundnut cake and fishmeal with no adverse effects on broiler performance. Raju et al. (2005) concluded that dietary inclusion of Spirulina at 0.05% can partially alleviate adverse effects of 300 ppm aflatoxin on growth rate and lymphoid organ weight of broiler chickens. It has also been reported that the amino acid pattern of Spirulina microalgae could be superior to the other vegetable feeds (e.g., soybean meal), and that they have a high amino acid digestibility (Alvarenga et al., 2011; Evans et al., 2015). In addition, Spirulina contains physiologically active substances such as carotenoid pigments, phycocyanin, polyunsaturated fatty acid, vitamins, macro- and micro-mineral elements, and many other chemical compounds (Becker, 2007; Eriksen, 2008; Maoka, 2011). These compounds confirm potential antimicrobial, antioxidant, and anti-inflammatory biological properties, or act as immune enhancers (Rathore et al., 2004; Rasool et al., 2009; Uyisenga et al., 2010; Langers et al., 2012; Abdel-Daim et al., 2013; Shokri et al., 2014). Therefore, the chemical composition and physiological functions of Spirulina seem to be involved in metabolism systems related to growth performance, and are likely to be the main cause of improvement of BWG, FCR, and EPEI in broiler chickens. GPx and SOD are generally thought to act as enzymatic free radical scavengers in cells (Abdel-Wahhab and Aly, 2005). In this study, GPx and SOD linearly increase in broiler chickens fed with Spirulina. Previous studies indicated that Spirulina contains antioxidants such as β-carotene, tocopherol, selenium, polypeptide pigment, or phenolic acids, some of which might contribute to antioxidant action together or with other various micronutrients (El-Desoky et al., 2013). Specifically, Spirulina is a rich source of phycocyanin, an antioxidant biliprotein pigment, which is related to other potent antioxidants (Khan et al., 2005). There is almost no information on antioxidant properties related to Spirulina in poultry, but there is some evidence of antioxidant activity from in vitro and several rat studies. Estrada et al. (2001) suggested that protean extracts of Spirulina had scavenging effects against hydroxyl radicals, with phycocyanin as the main component responsible for the antioxidant activity. Additionally, β-carotene and other carotenoids protect cells from oxidative stress by quenching singlet oxygen damage through a variety of mechanisms (Tinkler et al., 1994). Another probable cause is that increased levels of blood SOD and GPx confirmed in Spirulina groups may be associated with phenolic compounds. Many phenolic compounds including salicylic, trans-cinnamic, synaptic, chlorogenic, quinic, and caffeic acids present in Spirulina may also be responsible for its antioxidant activity, individually or synergistically (Miranda et al., 1998). Wu et al. (2005) suggested that Spirulina extract has stronger antioxidant capabilities than Chlorella, which is probably due to higher content of phenolic compounds (23.87 vs. 15.25 mg tannic acid equivalent/g of algae aqueous extract) and antioxidant capacity (ABTS assay: 19.74 vs. 4.60 μmol of Trolox equivalent/g of microalgae). Therefore, increased serum SOD and GPx concentrations in this study were likely due to chemical compounds such as phycocyanin, β-carotene, and phenolics in Spirulina, all relating to the antioxidant activities. In this study, the apparent total tract digestibilities of dry matter and nitrogen linearly increased in broilers fed with Spirulina diets, indicating that higher digestibility could be achieved with higher concentrations of Spirulina. The digestibility of Spirulina is not well documented, and the available studies on assimilation by poultry have not provided conclusive results. However, Mabeau and Fleurence (1993) confirmed that marine microalgae showed a high rate of protein degradation proteolytic enzymes such as pepsin, pancreatin, and pronase. Evans et al. (2015) reported that young broilers (21-d-old) had higher apparent ileal digestibility of glutamic acid, proline, glycine, alanine, methionine, leucine, and lysine when fed 6 to 21% Spirulina supplemented diets compared with broilers fed control diets. Furbeyre et al. (2017) reported that the total tract digestibility in pigs receiving 1% Spirulina and 1% Chlorella was greater for gross energy and tended to be greater for dry matter, organic matter, and neutral detergent fiber compared with control pigs. They also found that villus height at the jejunum was greater in pigs fed with Spirulina and Chlorella compared with control pigs. Microalgae are generally regarded as a viable protein source, with essential amino acid (EAA) composition meeting the Food and Agriculture Organization requirements, and are often on par with other protein sources, such as soybean and egg (Bleakley and Hayes, 2017). Increased digestibilities of dry matter and nitrogen in this study may be related to the high-quality protein containing balanced EAA. Therefore, the better digestibility of protein observed in Spirulina supplemented diets may be a result of better absorption, which enhanced growth in broiler chickens. The cecum plays an important role in preventing colonization of pathogens, detoxifying harmful substances, recycling nitrogen, microbial synthesis of vitamins, degradation of some carbohydrates, and absorbing additional nutrients (Coates et al., 1968; Clench and Mathias, 1995; Jorgensen et al., 1996). A previous study in broiler chickens also concluded that intestinal microbial-including cecum is highly associated with the production performance of broiler chickens (Jeong and Kim, 2014). Therefore, cecal microbial populations were investigated to evaluate Spirulina supplementation in broiler chickens. This study indicated that broiler chickens fed a Spirulina supplemented diet led to higher cecal Lactobacillus concentration, but there was no difference in the number of coliform bacteria. Some studies suggest that microalgae have potential antibacterial, antiviral, and antifungal activities. In vitro, de Mule et al. (1996) observed that methanolic and aqueous extracts of Spirulina inhibited the growth of Candida albicans by 17.6%, whereas Lactococcus lactis was promoted by the extract, with growth increasing from 7.5 to 14.7%. Kaushik and Chauhan (2008) demonstrated that the extracts of Spirulina have shown antibacterial effects by inhibiting the growth of harmful microorganisms, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumonia. The addition of dry Spirulina at 10 mg/mL into de Man, Rogosa, and Sharpe medium-promoted growth of Lactobacillus acidophilus by 186%, suggesting prebiotic potential of the microalgae (Bhowmik et al., 2009). Rania and Hala (2008) also suggested that Spirulina extract had antibacterial activities against E. coli because of the presence of alkaloids and lipopolysaccharides. In an in vivo study, feeding Chlorella microalgae resulted in increased Lactobacillus diversity in the crop or cecum or both of laying hens (Janczyk et al., 2009; Kang et al., 2013). To date, relatively few studies have investigated the antimicrobial activity of microalgae, including Spirulina and their extracts in poultry. We hypothesized that Spirulina supplementation would maintain the beneficial microbial population, and subsequently explain the improved digestibility and growth performance in this study. In regard to excreta noxious gas emission, the inclusion of Spirulina linearly reduced the excreta ammonia gas content as dietary levels of Spirulina increased. Excreta noxious gas emission of animals is associated with intestinal microflora, particularly harmful intestinal bacteria populations (Ferket et al., 2002). A previous study also suggested that lower excreta noxious gas content occurs when the microflora in the gastrointestinal tract of broiler chickens is manipulated (Jeong and Kim, 2014). Several other studies have also suggested that excreta noxious gas content is associated with nutrient digestibility (Yan et al., 2011; Jeong and Kim, 2014), because the increased digestibility may allow less substrate for microbial fermentation in the large intestine, which consequently decreases excreta noxious gas content. In our study, the Spirulina supplemented diet led to a better balanced microflora in the cecum and higher nutrient digestibility than that by control diet. Therefore, we suggest that the reason for reduction in excreta ammonia gas content may be the result of increased nitrogen digestibility and Lactobacillus populations in broiler chicken ceca. There is currently a lack of useful information regarding Spirulina in poultry, and previously reported functions of Spirulina in other species cannot be directly compared. However, findings from the current study support previous research (Yan et al., 2012) showing that dietary Chlorella supplementation decreased excreta ammonia gas emission in growing pigs. Venkataraman et al. (1994) showed that color pigmentation of skin, breast, and thigh muscles was deeper in broilers when substituting groundnut protein or fish meal with Spirulina up to 170 or 140 g/kg. Toyomizu et al. (2001) found that including Spirulina in broiler feeds influenced both the yellowness and redness of broiler meat. They reported that the increase of yellowness with dietary Spirulina content may be reflected in the common yellow pigment related to the accumulation of zeaxanthin within the meat. However, unlike previous reports that Spirulina has an effect on meat color, this study showed no differences in breast meat color of broiler chickens fed with Spirulina. Additionally, there were no differences in pH, cooking loss, WHC, or organ weights of broiler chickens fed with Spirulina. Drip loss causes nutrients and moisture to escape, and negatively affects chewiness of meat. Spirulina-supplemented diets showed a significant reduction of drip loss after 7 d of storage. However, reasons for this decrease remain unknown. Lu et al. (2014) suggested that dietary addition of antioxidants was effective in improving growth, moderately restored whole body antioxidant capability, and reduced drip loss. Asghar et al. (1989) and Attia et al. (2016) indicated that antioxidants preserve the functionality of membranes, thus improving their role as semipermeable barriers against exudative loss. According to Cheah et al. (1995), dietary antioxidant supplementation also prevents excessive drip loss from pale, soft, exudative muscle from stress-susceptible pigs. This action seems to result from decreased membrane phospholipase activity through higher antioxidant content of the tissue membranes, because drip loss occurs when phospholipids in the intracellular membrane are oxidized and the membrane becomes weak. In this study, decreased drip loss due to the inclusion of dietary Spirulina is, presumably, related to the delayed oxidation of the cell membrane. Improvement was probably due to the positive effect of bioactive compounds, including antioxidants of Spirulina, on the integrity of muscle fibers; thus, enhancing their capability to retain water (Dal Bosco et al., 2014). The findings of our study support previous research, which determined that Spirulina could affect meat quality by decreasing the drip loss of Japanese quail meats (Cheong et al., 2016). Further studies are needed to determine the mechanisms involved in the association between Spirulina and drip loss of meat. In conclusion, supplementation with Spirulina improved BWG, FCR, and EPEI; increased antioxidant enzyme activity, dry matter and nitrogen digestibilities; increased cecal Lactobacillus populations; and decreased excreta ammonia gas emission in broiler chickens. For breast meat quality, Spirulina decreased drip loss after 7 d of storage. Therefore, Spirulina microalgae at 1.0% diet might provide a good alternative to improve broiler chicken production. Acknowledgements This research was financially supported by the Ministry of Trade, Industry, and Energy (MOTIE), Korea, under the “Regional Specialized Industry Development Program”(reference number R0005540) supervised by the Korea Institute for Advancement of Technology (KIAT). REFERENCES Abdel-Daim M. M. , Abuzead S. M. , Halawa S. M. . 2013 . Protective role of Spirulina platensis against acute deltamethrin-induced toxicity in rats . PLoS One . 8 : e72991 . Google Scholar CrossRef Search ADS PubMed Abdel-Wahhab M. A. , Aly S. E. . 2005 . Antioxidant property of Nigella sativa (black cumin) and Syzygium aromaticum (clove) in rats during aflatoxicosis . J. Appl. Toxicol. 25 : 218 – 223 . Google Scholar CrossRef Search ADS PubMed Al-Batshan H. A. , Al-Mufarrej S. I. , Al-Homaidan A. A. , Qureshi M. A. . 2001 . Enhancement of chicken macrophage phagocytic function and nitrite production by dietary Spirulina platensis . Immunopharmacol. Immunotoxicol. 23 : 281 – 289 . Google Scholar CrossRef Search ADS PubMed Alvarenga R. R. , Rodrigues P. B. , Cantarelli V. , Zangeronimo M. G. , Da Silva Junior J. W. , Da Silva L. R. , Dos Santos L. M. , Pereira L. J. . 2011 . Energy values and chemical composition of Spirulina (Spirulina platensis) evaluated with broilers . Braz. J. Anim. Sci. 40 : 992 – 996 . AOAC . 2000 . Official Method of Analysis . 17th ed . Assoc. Off. Anal. Chem. , Washington, DC . PubMed PubMed Asghar A. , Lin C. F. , Gray J. I. , Buckley D. J. , Booren A. M. , Crackel R. L. , Flegal C. J. . 1989 . Influence of oxidised oil and antioxidant supplementation on membrane bound lipid stability in broiler meat . Br. Poult. Sci. 30 : 815 – 823 . Google Scholar CrossRef Search ADS PubMed Attia Y. A. , Al-Harthi M. A. , Korish M. M. , Shiboob M. M. . 2016 . Evaluation of the broiler meat quality in the retail market: Effects of type and source of carcasses . Rev. Mex. Cienc. Pecu. 7 : 321 – 339 . Becker E. W. 2007 . Micro-algae as a source of protein . Biotechnol. Adv. 25 : 207 – 210 . Google Scholar CrossRef Search ADS PubMed Bhavisha R. , Parula P. . 2010 . Spirulina: potential clinical therapeutic application . J. Pharm. Res. 3 : 1726 – 1732 . Bhowmik D. , Dubey J. , Mehra S. . 2009 . Probiotic efficiency of Spirulina platensis: stimulating growth of lactic acid bacteria . Am. Eurasian J. Agric. Environ. Sci. 6 : 546 – 549 . Bleakley S. , Hayes M. . 2017 . Algal proteins: extraction, application, and challenges concerning production . Foods 26 : 6 . pii: E33 . Cheah K. S. , Cheah A. M. , Krausgrill D. I. . 1995 . Effect of dietary supplementation of vitamin E on pig meat quality . Meat Sci. 39 : 255 – 264 . Google Scholar CrossRef Search ADS PubMed Cheong D. S. W. , Kasim A. , Sazili A. Q. , Omar H. , Teoh J. Y. . 2016 . Effect of supplementing Spirulina on live performance, carcass composition and meat quality of Japanese quail . Walailak J. Sci. Tech. 13 : 77 – 84 . Clench M. H. , Mathias J. R. . 1995 . The avian cecum: a review . Wilson Bull . 107 : 93 – 121 . Coates M. E. , Ford J. E. , Harrison G. F. . 1968 . Intestinal synthesis of vitamins of the B complex in chicks . Br. J. Nutr. 22 : 493 – 498 . Google Scholar CrossRef Search ADS PubMed Dal Bosco A. , Gerencsér Z. , Szendrő Z. , Mugnai C. , Cullere M. , Kovács M. , Ruggeri S. , Mattioli S. , Castellini C. , Dalle Zotte A. . 2014 . Effect of dietary supplementation of Spirulina (Arthrospira platensis) and Thyme (Thymus vulgaris) on rabbit meat appearance, oxidative stability and fatty acid profile during retail display . Meat Sci. 96 : 114 – 119 . Google Scholar CrossRef Search ADS PubMed de Mule´ M. C. Z. , de Caire G. Z. , de Cano M. S. . 1996 . Bioactive substances from Spirulina platensis (Cyanobacteria) . Phyton . 58 : 93 – 96 . El-Desoky G. E. , Bashandy S. A. , Alhazza I. M. , Al-Othman Z. A. , Aboul-Soud M. A. , Yusuf K. . 2013 . Improvement of mercuric chloride-induced testis injuries and sperm quality deteriorations by Spirulina platensis in rats . PLoS One . 8 : e59177 . Google Scholar CrossRef Search ADS PubMed Eriksen N. T. 2008 . Production of phycocyanin—a pigment with applications in biology, biotechnology, foods and medicine . Appl. Microbiol. Biotechnol. 80 : 1 – 14 . Google Scholar CrossRef Search ADS PubMed Evans A. M. , Smith D. L. , Moritz J. S. . 2015 . Effects of algae incorporation into broiler starter diet formulations on nutrient digestibility and 3 to 21 d bird performance . J. Appl. Poult. Res. 24 : 206 – 214 . Google Scholar CrossRef Search ADS Estrada J. P. , Bescós P. B. , Del Fresno A. V. . 2001 . Antioxidant activity of different fractions of Spirulina platensis protean extract . Il Farmaco . 56 : 497 – 500 . Google Scholar CrossRef Search ADS PubMed Ferket P. R. , van Heugten E. , van Kempen T. A. T. G. , Angel R. . 2002 . Nutritional strategies to reduce environmental emissions from nonruminants . J. Anim. Sci. 80 : E168 – E182 . Google Scholar CrossRef Search ADS Furbeyre H. , van Milgen J. , Mener T. , Gloaguen M. , Labussiere E. . 2017 . Effects of dietary supplementation with freshwater microalgae on growth performance, nutrient digestibility and gut health in weaned piglets . Animal . 11 : 183 – 192 . Google Scholar CrossRef Search ADS PubMed Janczyk P. , Halle B. , Souffrant W. B. . 2009 . Microbial community composition of the crop and ceca contents of laying hens fed diets supplemented with Chlorella vulgaris . Poult. Sci. 88 : 2324 – 2332 . Google Scholar CrossRef Search ADS PubMed Jeong J. S. , Kim I. H. . 2014 . Effect of Bacillus subtilis C-3102 spores as a probiotic feed supplement on growth performance, noxious gas emission, and intestinal microflora in broilers . Poult. Sci. 93 : 3097 – 3103 . Google Scholar CrossRef Search ADS PubMed Jorgensen H. , Zhao X. Q. , Knudsen K. E. , Eggum B. O. . 1996 . The influence of dietary fibre source and level on the development of the gastrointestinal tract, digestibility and energy metabolism in broiler chickens . Br. J. Nutr. 75 : 379 – 395 . Google Scholar CrossRef Search ADS PubMed Joventino I. P. , Alves H. G. , Neves L. C. , Pinheiro-Joventino F. , Leal L. K. , Neves S. A. , Ferreira F. V. , Brito G. A. , Viana G. B. . 2012 . The microalga Spirulina platensis presents anti-inflammatory action as well as hypoglycemic and hypolipidemic properties in diabetic rats . J. Complement. Integr. Med. 9 : 17 . Google Scholar CrossRef Search ADS Kang H. K. , Salim H. M. , Akter N. , Kim D. W. , Kim J. H. , Bang H. T. , Kim M. J. , Na J. C. , Hwangbo J. , Choi H. C. , Suh O. S. . 2013 . Effect of various forms of dietary Chlorella supplementation on growth performance, immune characteristics, and intestinal microflora population of broiler chickens . J. Appl. Poult. Res. 22 : 100 – 108 . Google Scholar CrossRef Search ADS Kaushik P. , Chauhan A. . 2008 . In vitro antibacterial activity of laboratory grown culture of Spirulina platensis . Indian J. Microbiol. 48 : 348 – 352 . Google Scholar CrossRef Search ADS PubMed Khan Z. , Bhadouria P. , Bisen P. S. . 2005 . Nutritional and therapeutic potential of Spirulina . Curr. Pharm. Biotechnol. 6 : 373 – 379 . Google Scholar CrossRef Search ADS PubMed Korean Feeding Standards for Poultry . 2012 . National Institute of Animal Science , RDA , Suwon, Republic of Korea . Kristensen L. , Purslow P. P. . 2001 . The effect of ageing on the water-holding capacity of pork: role of cytoskeletal proteins . Meat Sci. 58 : 17 – 23 . Google Scholar CrossRef Search ADS PubMed Kulshreshtha A. , Jarouliya U. , Bhadauriya P. , Prasad G. B. K. S. , Bisen P. S. . 2008 . Spirulina in health care management . Curr. Pharm. Biotechnol. 9 : 400 – 405 . Google Scholar CrossRef Search ADS PubMed Langers I. , Renoux V. M. , Thiry M. , Delvenne P. , Jacobs N. . 2012 . Natural killer cells: role in local tumor growth and metastasis . Biologics . 6 : 73 – 82 . Google Scholar PubMed Lu T. , Harper A. F. , Zhao J. , Dalloul R. A. . 2014 . Effects of a dietary antioxidant blend and vitamin E on growth performance, oxidative status, and meat quality in broiler chickens fed a diet high in oxidants . Poult. Sci. 93 : 1649 – 1657 . Google Scholar CrossRef Search ADS PubMed Mabeau S. , Fleurence J. . 1993 . Seaweed in food products: biochemical and nutritional aspects . Trends Food Sci. Technol. 4 : 103 – 107 . Google Scholar CrossRef Search ADS Meineri G. , Ingravalle F. , Radice E. , Aragno M. , Peiretti P. G. , 2009 : Effects of high fat diets and Spirulina platensis supplementation in New Zealand white rabbits . J. Anim. Vet. Adv. 8 : 2735 – 2744 . Maoka T. 2011 . Carotenoids in marine animals . Marine Drugs . 9 : 278 – 293 . Google Scholar CrossRef Search ADS PubMed Miranda M. S. , Cintra R. G. , Barros S. B. M. , Filho J. M. . 1998 . Antioxidant activity of the microalga Spirulina maxima . Braz. J. Med. Biol. Res. 31 : 1075 – 1079 . Google Scholar CrossRef Search ADS PubMed National research council (NRC) . 1994 . Nutrient Requirements of Poultry . 9th rev. ed . National Academy Press , Washington DC . Raju M. V. L. N. , Rao S. V. , Radhika K. , Chawak M. M. . 2005 . Dietary supplementation of Spirulina and its effects on broiler chicken exposed to aflatoxicosis . Indian J. Poult. Sci. 40 : 36 – 40 . Rania M. A. , Hala M. T. . 2008 . Antibacterial and antifungal activity of cyanobacteria and green microalgae. Evaluation of medium components by placket-burman design for antimicrobial activity of Spirulina platensis . Global J. Biotechnol. Biochem. 3 : 22 – 31 . Rasool M. K. , Sabina E. P. , Nithya P. , Lavanya K. . 2009 . Suppressive effect of Spirulina fusiformis in relation to lysosomal acid hydrolases, lipid peroxidation, antioxidant status, and inflammatory mediator TNF-alpha on experimental gouty arthritis in mice . Orient. Pharm. Exp. Med. 9 : 164 – 173 . Google Scholar CrossRef Search ADS Rathore N. K. , Singh S. , Singh V. . 2004 . Spirulina as immuno-enhancer and biomodulator . J. Immunol. Immunopathol. 6 : 1 – 10 . Saxena P. N. , Ahmad M. R. , Shyam R. , Amla D. V. . 1983 . Cultivation of Spirulina in sewage for poultry feed . Experientia 39 : 1077 – 1083 . Google Scholar CrossRef Search ADS Shields R. J. , Lupatsch I. . 2012 . Algae for aquaculture and animal feeds . J. Anim. Sci. 21 : 23 – 37 . Shokri H. , Khosravi A. , Taghavi M. . 2014 . Efficacy of Spirulina platensis on immune functions in cancer mice with systemic candidiasis . J. Mycol. Res. 1 : 7 – 13 . Sujatha T. , Narahari D. . 2011 . Effect of designer diets on egg yolk composition of ‘White Leghorn’ hens . J. Food Sci. Technol. 48 : 494 – 497 . Google Scholar CrossRef Search ADS PubMed Tinkler J. H. , Bohm F. , Schalch W. , Truscott T. G. . 1994 . Dietary carotenoids protect human cells from damage . J. Photochem. Photobiol. 26 : 283 – 285 . Google Scholar CrossRef Search ADS Toyomizu M. , Sato K. , Taroda H. , Kato T. , Akiba Y. . 2001 . Effects of dietary Spirulina on meat colour in muscle of broiler chickens . Br. Poult. Sci. 42 : 197 – 202 . Google Scholar CrossRef Search ADS PubMed Uyisenga J. P. , Nzayino P. , Seneza R. , Hishamunda L. , Uwantege K. , Gasana N. , Emmanuel S. B. . 2010 . In vitro study of antibacterial and antifungal activity of Spirulina platensis . Int. J. Ecol. Dev. 16 : 80 – 88 . Venkataraman L. V. , Somasekaran T. , Becker E. W. . 1994 . Replacement value of blue-green alga (Spirulina platensis) for fishmeal and a vitamin-mineral premix for broiler chicks . Br. Poult. Sci. 35 : 373 – 381 . Google Scholar CrossRef Search ADS PubMed Wu L. C. , Ho J. A. A. , Shieh M. C. , Lu I. W. . 2005 . Antioxidant and antiproliferative activities of Spirulina and Chlorella water extracts . J. Agric. Food Chem. 53 : 4207 – 4212 . Google Scholar CrossRef Search ADS PubMed Yan L. , Meng Q. W. , Kim I. H. . 2011 . The effect of an herb extract mixture on growth performance, nutrient digestibility, blood characteristics and fecal noxious gas content in growing pigs . Livestock Sci. 141 : 143 – 147 . Google Scholar CrossRef Search ADS Yan L. , Lim S. U. , Kim I. H. . 2012 . Effect of fermented chlorella supplementation on growth performance, nutrient digestibility, blood characteristics, fecal microbial and fecal noxious gas content in growing pigs . Asian Australas. J. Anim. Sci 25 : 1742 – 1747 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the term of oxford University Press, standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Effect of dietary Spirulina (Arthrospira) platensis on the growth performance, antioxidant enzyme activity, nutrient digestibility, cecal microflora, excreta noxious gas emission, and breast meat quality of broiler chickens

Poultry Science , Volume Advance Article (7) – Jun 22, 2018

Loading next page...
 
/lp/ou_press/effect-of-dietary-spirulina-arthrospira-platensis-on-the-growth-e0iLIeHSmM
Publisher
Oxford University Press
Copyright
© 2018 Poultry Science Association Inc.
ISSN
0032-5791
eISSN
1525-3171
D.O.I.
10.3382/ps/pey093
Publisher site
See Article on Publisher Site

Abstract

ABSTRACT This study examined the effects of dietary Spirulina (Arthrospira) platensis supplementation on growth performance, antioxidant enzyme activity, nutrient digestibility, cecal microflora, excreta noxious gas emission, organ weight and breast meat quality in broiler chickens. In total, 800 Ross 308 male broiler chickens (1-d-old) were randomly divided into 5 dietary treatments with 10 replicate cages (16 birds/replicate) per treatment for 5 wk. The dietary treatments were a control basal diet without Spirulina or with 0.25, 0.5, 0.75, or 1.0% Spirulina. Body weight gain, feed conversion, and/or European production efficiency index improved linearly with supplementation of Spirulina during d 8 to 21, 22 to 35, and overall d 1 to 35 (P < 0.05). Dietary Spirulina supplementation caused a significant increase in the serum enzyme activity of superoxide dismutase and glutathione peroxidase (linear, P < 0.05). Apparent total tract digestibility of dry matter and nitrogen showed a linear increase in Spirulina supplementation (P < 0.05). Cecal Lactobacillus count linearly increased and excreta ammonia gas emission linearly decreased, as dietary Spirulina supplementation increased (P < 0.05). There were no significant effects on relative organ weight and breast meat quality of broilers fed with Spirulina diets; however, 7 d drip loss linearly decreased in treatment groups fed with Spirulina (P < 0.05). These results indicate that adding Spirulina to the diet of broilers can improve antioxidant enzyme activity, dry matter and nitrogen digestibility, cecal Lactobacillus population, excreta ammonia gas emission, and 7 d drip loss of breast meat. In addition, dietary inclusion of 1.0% Spirulina powder might provide a good alternative to improve broiler chicken production. INTRODUCTION Microalgae are attracting attention as the future clean energy and industrial material resources such as food, drug, cosmetics, and organic fertilizers because they can be mass-produced in a short time in various environments. In addition, Chlorella, Schizochytrium, and Spirulina are recognized as renewable substitutes for conventional protein sources (e.g., soybean meal, fish meal, rice bran) in aquaculture or animal feed because of their nutritional importance (Shields and Lupatsch, 2012). Spirulina (Arthrospira) platensis is a filamentous blue-green microalgae (cyanobacteria) generally regarded as a rich source of high quality protein, vitamins (particularly vitamin B12 and provitamin β-carotene), minerals, essential fatty acids, essential amino acids, pigments, and phenolic acids (Kulshreshtha et al., 2008; Bhavisha and Parula, 2010; Joventino et al., 2012). Many research studies have shown that Spirulina has antioxidant, immunomodulatory, anti-inflammatory, antiviral, and antimicrobial activity in various experimental animals (Rasool et al., 2009; Uyisenga et al., 2010; Langers et al., 2012; Abdel-Daim et al., 2013; Shokri et al., 2014). Recently, there has been a growing interest in its application in animals for its antioxidant activity, growth-promoting role, and immunomodulatory effects. These positive effects of Spirulina in the body may ultimately lead to improved animal productivity. For example, in feeding trials with livestock animals, Spirulina has been found to increase growth rate, nutrient utilization, disease resistance, egg quality, and carcass quality in poultry, pigs, and rabbit (Al-Batshan et al., 2001; Meineri et al., 2009; Sujatha and Narahari, 2011; Evans et al., 2015). However, present knowledge of broiler chicken response to dietary Spirulina supplementation is relatively unknown. The purpose of this study was to investigate the effect of Spirulina microalgae as a feed ingredient source in broiler chicken diets. Feeding experiments with broiler chickens were conducted to assess nutritional physiological properties, as well as to investigate effects on growth performance. MATERIALS AND METHODS Experimental protocols describing the management and care of animals were reviewed and approved by the Animal Care and Use Committee of Dankook University (Approval No. DK-1–1642), Republic of Korea. Animals and Housing A total of 800 male broiler chickens (1-d-old, Ross 308) were obtained from a commercial hatchery. Broiler chickens of similar body weight (41.5 ± 0.5 g) were randomly distributed into 5 groups (160 birds in 10 cages per treatment, 16 birds/cage). Broilers were housed in a temperature-controlled room with 3 floors of stainless steel battery cages (124 cm-width × 64 cm-length × 40 cm-height), which allowed free access to feed and water during the experimental period. They were kept in a room with controlled temperature and light regimen of 22L:2D for the entire experimental period. The environmental temperature was maintained at 33°C for the first week and then gradually reduced to 20°C by the fifth week. Relative humidity was gradually increased from 60% (d 1 to 21) to 70% (d 22 to 35). Diets Broilers were fed a corn/soybean-based basal diet for 35 d divided in 3 phases: Phase 1 (d 1 to 7), Phase 2 (d 8 to 21), and Phase 3 (d 22 to 35) (Table 1). The experimental diets, in mash form, were formulated to meet and exceed the nutrients requirements of NRC (1994) and Korean Feeding Standard for Poultry (2012). The dietary treatments were a control basal diet without Spirulina or with 0.25, 0.5, 0.75, or 1.0% Spirulina. A commercially available freeze-dried Spirulina powder was provided by a private company (NeoEnBiz Co., Bucheon, Republic of Korea) and supplemented to the basal diet, at the expense of soybean meal. Table 2 shows the nutrient composition of the freeze-dried Spirulina powder. Table 1. Ingredient composition of experimental diets (as-fed basis). Ingredients, % Phase 1 (d 1 to 7) Phase 2 (d 8 to 21) Phase 3 (d 22 to 35) Corn 35.22 39.82 37.56 Soybean meal 37.26 32.06 28.64 Wheat 15.00 15.00 20.00 Animal fat 4.15 5.07 6.61 Corn gluten meal 3.78 3.86 3.24 Monodicalcium phosphate 1.22 1.08 0.90 Limestone 1.80 1.64 1.61 Salt 0.36 0.36 0.63 Choline-chloride 0.14 0.13 0.12 L-Lysine 0.39 0.37 0.36 DL-Methionine 0.27 0.24 0.24 L-Threonine 0.09 0.07 0.07 Vit. Mix1 0.12 0.11 0.10 Min. Mix2 0.10 0.10 0.10 Phytase 0.10 0.10 0.10 Calculated values  ME, kcal/kg 3,000 3,100 3,200  CP, % 23.00 22.00 20.00  Dig. Lys, % 1.32 1.10 1.00  Dig. Met, % 0.52 0.50 0.38  Dig. Met + Cys, % 1.05 0.90 0.72  Dig. Thr, % 0.94 0.80 0.74  Dig. Iso, % 0.95 0.80 0.73  Dig. Val, % 1.09 0.90 0.82  Dig. Arg, % 1.45 1.25 1.10  Ca, % 1.00 1.00 0.90  Total P, % 0.80 0.80 0.75  Available P, % 0.45 0.45 0.35  Na, % 0.20 0.20 0.15 Analyzed values  CP, % 23.87 21.87 20.28  Lys, % 1.42 1.27 1.17  Met, % 0.64 0.58 0.56  Ca, % 1.05 0.92 0.85  P, % 0.74 0.76 0.72 Ingredients, % Phase 1 (d 1 to 7) Phase 2 (d 8 to 21) Phase 3 (d 22 to 35) Corn 35.22 39.82 37.56 Soybean meal 37.26 32.06 28.64 Wheat 15.00 15.00 20.00 Animal fat 4.15 5.07 6.61 Corn gluten meal 3.78 3.86 3.24 Monodicalcium phosphate 1.22 1.08 0.90 Limestone 1.80 1.64 1.61 Salt 0.36 0.36 0.63 Choline-chloride 0.14 0.13 0.12 L-Lysine 0.39 0.37 0.36 DL-Methionine 0.27 0.24 0.24 L-Threonine 0.09 0.07 0.07 Vit. Mix1 0.12 0.11 0.10 Min. Mix2 0.10 0.10 0.10 Phytase 0.10 0.10 0.10 Calculated values  ME, kcal/kg 3,000 3,100 3,200  CP, % 23.00 22.00 20.00  Dig. Lys, % 1.32 1.10 1.00  Dig. Met, % 0.52 0.50 0.38  Dig. Met + Cys, % 1.05 0.90 0.72  Dig. Thr, % 0.94 0.80 0.74  Dig. Iso, % 0.95 0.80 0.73  Dig. Val, % 1.09 0.90 0.82  Dig. Arg, % 1.45 1.25 1.10  Ca, % 1.00 1.00 0.90  Total P, % 0.80 0.80 0.75  Available P, % 0.45 0.45 0.35  Na, % 0.20 0.20 0.15 Analyzed values  CP, % 23.87 21.87 20.28  Lys, % 1.42 1.27 1.17  Met, % 0.64 0.58 0.56  Ca, % 1.05 0.92 0.85  P, % 0.74 0.76 0.72 1Provided per kg of complete diet: 11,025 IU of vitamin A; 1,103 IU of vitamin D3; 44 IU of vitamin E; 4.4 mg of vitamin K; 8.3 mg of riboflavin; 50 mg of niacin; 4 mg of thiamine; 29 mg of d-pantothenic; 166 mg of choline; 33 μg of vitamin B12. 2Provided per kg of complete diet: 12 mg of Cu (as CuSO4.5H2O); 85 mg of Zn (as ZnSO4); 8 mg of Mn (as MnO2); 0.28 mg of I (as KI); 0.15 mg of Se (as Na2SeO3.5H2O). View Large Table 1. Ingredient composition of experimental diets (as-fed basis). Ingredients, % Phase 1 (d 1 to 7) Phase 2 (d 8 to 21) Phase 3 (d 22 to 35) Corn 35.22 39.82 37.56 Soybean meal 37.26 32.06 28.64 Wheat 15.00 15.00 20.00 Animal fat 4.15 5.07 6.61 Corn gluten meal 3.78 3.86 3.24 Monodicalcium phosphate 1.22 1.08 0.90 Limestone 1.80 1.64 1.61 Salt 0.36 0.36 0.63 Choline-chloride 0.14 0.13 0.12 L-Lysine 0.39 0.37 0.36 DL-Methionine 0.27 0.24 0.24 L-Threonine 0.09 0.07 0.07 Vit. Mix1 0.12 0.11 0.10 Min. Mix2 0.10 0.10 0.10 Phytase 0.10 0.10 0.10 Calculated values  ME, kcal/kg 3,000 3,100 3,200  CP, % 23.00 22.00 20.00  Dig. Lys, % 1.32 1.10 1.00  Dig. Met, % 0.52 0.50 0.38  Dig. Met + Cys, % 1.05 0.90 0.72  Dig. Thr, % 0.94 0.80 0.74  Dig. Iso, % 0.95 0.80 0.73  Dig. Val, % 1.09 0.90 0.82  Dig. Arg, % 1.45 1.25 1.10  Ca, % 1.00 1.00 0.90  Total P, % 0.80 0.80 0.75  Available P, % 0.45 0.45 0.35  Na, % 0.20 0.20 0.15 Analyzed values  CP, % 23.87 21.87 20.28  Lys, % 1.42 1.27 1.17  Met, % 0.64 0.58 0.56  Ca, % 1.05 0.92 0.85  P, % 0.74 0.76 0.72 Ingredients, % Phase 1 (d 1 to 7) Phase 2 (d 8 to 21) Phase 3 (d 22 to 35) Corn 35.22 39.82 37.56 Soybean meal 37.26 32.06 28.64 Wheat 15.00 15.00 20.00 Animal fat 4.15 5.07 6.61 Corn gluten meal 3.78 3.86 3.24 Monodicalcium phosphate 1.22 1.08 0.90 Limestone 1.80 1.64 1.61 Salt 0.36 0.36 0.63 Choline-chloride 0.14 0.13 0.12 L-Lysine 0.39 0.37 0.36 DL-Methionine 0.27 0.24 0.24 L-Threonine 0.09 0.07 0.07 Vit. Mix1 0.12 0.11 0.10 Min. Mix2 0.10 0.10 0.10 Phytase 0.10 0.10 0.10 Calculated values  ME, kcal/kg 3,000 3,100 3,200  CP, % 23.00 22.00 20.00  Dig. Lys, % 1.32 1.10 1.00  Dig. Met, % 0.52 0.50 0.38  Dig. Met + Cys, % 1.05 0.90 0.72  Dig. Thr, % 0.94 0.80 0.74  Dig. Iso, % 0.95 0.80 0.73  Dig. Val, % 1.09 0.90 0.82  Dig. Arg, % 1.45 1.25 1.10  Ca, % 1.00 1.00 0.90  Total P, % 0.80 0.80 0.75  Available P, % 0.45 0.45 0.35  Na, % 0.20 0.20 0.15 Analyzed values  CP, % 23.87 21.87 20.28  Lys, % 1.42 1.27 1.17  Met, % 0.64 0.58 0.56  Ca, % 1.05 0.92 0.85  P, % 0.74 0.76 0.72 1Provided per kg of complete diet: 11,025 IU of vitamin A; 1,103 IU of vitamin D3; 44 IU of vitamin E; 4.4 mg of vitamin K; 8.3 mg of riboflavin; 50 mg of niacin; 4 mg of thiamine; 29 mg of d-pantothenic; 166 mg of choline; 33 μg of vitamin B12. 2Provided per kg of complete diet: 12 mg of Cu (as CuSO4.5H2O); 85 mg of Zn (as ZnSO4); 8 mg of Mn (as MnO2); 0.28 mg of I (as KI); 0.15 mg of Se (as Na2SeO3.5H2O). View Large Table 2. Nutrient composition of freeze-dried Spirulina powder at −50°C. Items % Moisture 5.4 Protein 56.7 Fiber 0.01 Ash 7.8 Amino acid  Aspartate 6.2  Threonine 3.2  Serine 3.3  Glutamate 8.6  Proline 2.4  Glycine 3.1  Alanine 4.8  Valine 3.3  Methionine 1.2  Isoleucine 2.6  Leucine 5.2  Tyrosine 2.4  Phenylalanine 2.7  Lysine 3.1  Histidine 0.9  Arginine 3.7 Fatty acid  Lauric acid (C12:0) 0.4  Myristic acid (C14:0) 0.7  Palmitic acid (C16:0) 15.5  Stearic acid (C18:0) 3.2  Arachidic acid (C20:0) 0.4  Behenic acid (C22:0) 0.4  Lignoceric acid (C24:0) 0.4  Palmitoleic acid (C16:1) 0.7  Oleic acid (C18:1) 32.5  Linoleic acid (C18:2n−6) 22.7  α-Linolenic acid (C18:3n−3) 21.4  Gadoleic acid (C20:1) 0.6 Items % Moisture 5.4 Protein 56.7 Fiber 0.01 Ash 7.8 Amino acid  Aspartate 6.2  Threonine 3.2  Serine 3.3  Glutamate 8.6  Proline 2.4  Glycine 3.1  Alanine 4.8  Valine 3.3  Methionine 1.2  Isoleucine 2.6  Leucine 5.2  Tyrosine 2.4  Phenylalanine 2.7  Lysine 3.1  Histidine 0.9  Arginine 3.7 Fatty acid  Lauric acid (C12:0) 0.4  Myristic acid (C14:0) 0.7  Palmitic acid (C16:0) 15.5  Stearic acid (C18:0) 3.2  Arachidic acid (C20:0) 0.4  Behenic acid (C22:0) 0.4  Lignoceric acid (C24:0) 0.4  Palmitoleic acid (C16:1) 0.7  Oleic acid (C18:1) 32.5  Linoleic acid (C18:2n−6) 22.7  α-Linolenic acid (C18:3n−3) 21.4  Gadoleic acid (C20:1) 0.6 View Large Table 2. Nutrient composition of freeze-dried Spirulina powder at −50°C. Items % Moisture 5.4 Protein 56.7 Fiber 0.01 Ash 7.8 Amino acid  Aspartate 6.2  Threonine 3.2  Serine 3.3  Glutamate 8.6  Proline 2.4  Glycine 3.1  Alanine 4.8  Valine 3.3  Methionine 1.2  Isoleucine 2.6  Leucine 5.2  Tyrosine 2.4  Phenylalanine 2.7  Lysine 3.1  Histidine 0.9  Arginine 3.7 Fatty acid  Lauric acid (C12:0) 0.4  Myristic acid (C14:0) 0.7  Palmitic acid (C16:0) 15.5  Stearic acid (C18:0) 3.2  Arachidic acid (C20:0) 0.4  Behenic acid (C22:0) 0.4  Lignoceric acid (C24:0) 0.4  Palmitoleic acid (C16:1) 0.7  Oleic acid (C18:1) 32.5  Linoleic acid (C18:2n−6) 22.7  α-Linolenic acid (C18:3n−3) 21.4  Gadoleic acid (C20:1) 0.6 Items % Moisture 5.4 Protein 56.7 Fiber 0.01 Ash 7.8 Amino acid  Aspartate 6.2  Threonine 3.2  Serine 3.3  Glutamate 8.6  Proline 2.4  Glycine 3.1  Alanine 4.8  Valine 3.3  Methionine 1.2  Isoleucine 2.6  Leucine 5.2  Tyrosine 2.4  Phenylalanine 2.7  Lysine 3.1  Histidine 0.9  Arginine 3.7 Fatty acid  Lauric acid (C12:0) 0.4  Myristic acid (C14:0) 0.7  Palmitic acid (C16:0) 15.5  Stearic acid (C18:0) 3.2  Arachidic acid (C20:0) 0.4  Behenic acid (C22:0) 0.4  Lignoceric acid (C24:0) 0.4  Palmitoleic acid (C16:1) 0.7  Oleic acid (C18:1) 32.5  Linoleic acid (C18:2n−6) 22.7  α-Linolenic acid (C18:3n−3) 21.4  Gadoleic acid (C20:1) 0.6 View Large Growth Performance Body weight (BW) and feed intake (FI) per cage were recorded on d 7, 21, and 35, and the feed conversion ratio (FCR) was calculated based on feed intake divided by body weight gain (BWG). Mortality was recorded daily, and percentage mortality was calculated throughout the study. The European production efficiency index (EPEI) was calculated with following formula. EPEI = (BW/d × survival rate/FCR × 10). Antioxidant Enzyme Activity Analysis At the end of the experiment (35 d), blood samples were collected from the left wing vein into K3EDTA vacuum tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ), and stored at 4°C. For serum analysis, approximately 3 mL of blood samples were centrifuged at 4,000 × g for 15 min at 4°C, after which the serum was separated. Antioxidant enzyme activities, superoxide dismutase (SOD), and glutathione peroxidase (GPx) in serum were measured using a commercial kit from Cayman Chemical Company (Cayman Chemical Co., Ann Arbor, MI, USA), according to the manufacturer's instructions. Apparent Total Tract Digestibility To determine the apparent total tract digestibility, 0.2% chromic oxide was added to the experimental diets 4 d prior to the collection period. Excreta were collected daily for the last 3 d of the experiment, and placed into a 60°C oven for 72 h. After drying, excreta were pulverized to pass through a 1-mm screen, and dry matter and nitrogen in diets and excreta were analyzed (methods 934.01 and 968.06; AOAC, 2000). Chromium concentration was determined by atomic absorption spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan). The equation for calculating digestibility was as follows: digestibility (%) = (1 – ((Nf × Cd)/(Nd × Cf))) × 100, where Nf = nutrient concentration in feces (% DM), Nd = nutrient concentration in diet (% DM), Cf = chromium concentration in feces (% DM), and Cd = chromium concentration in diet (% DM). Cecal Microflora Population One gram of cecal sample was blended with 9 mL of sterile peptone water and mixed for 1 min on a vortex stirrer. Viable counts of bacteria in the cecal samples were conducted by plating serial 10-fold dilutions (10−1 to 10−8) onto Lactobacilli MRS agar (Difco Laboratories, Detroit, MI, USA) plates and MacConkey agar (Difco Laboratories, Detroit, MI, USA) plates to isolate Lactobacillus spp. and coliform bacteria, respectively. The lactobacilli agar plates were then incubated for 48 h at 37°C under anaerobic conditions. The MacConkey agar plates were incubated for 24 h at 37°C under aerobic conditions. After the incubation periods, colonies of the respective bacteria were counted and expressed as the logarithm of colony-forming units per gram (log10 CFU/g). Excreta Noxious Gas Emission During the last 3 d of the experiment, fresh excreta samples were collected from each replication for analyzing ammonia, hydrogen sulfide, and total mercaptan. The excreta samples were kept in 3 L sealed plastic containers for 5 d at room temperature (24°C). After the fermentation period, a Gastec (model GV-100) gas sampling pump was utilized for gas detection (Gastec Corp., Tokyo, Japan). Concentrations of ammonia, hydrogen sulfide, and total mercaptan were measured within the scope of 5.0 to 100.0 (No. 3La, detector tube; Gastec Corp.), 2.0 to 20.0 (No. 4LK, detector tube; Gastec Corp.), and 0.5 to 120.0 (No.70 and 70-L, detector tubes; Gastec Corp.) ppm. The adhesive plaster was punctured, and 100 mL of headspace air was sampled at approximately 3 cm above the excreta. Breast Meat Quality and Relative Organ Weight Color values of breast meat were measured in 3 replicates using a Minolta colorimeter (CR-300, Tokyo, Japan) calibrated with a standard white plate and recorded as L*, a*, and b* values for lightness, redness, and yellowness, respectively. The pH values of raw breast meat were measured using a pH meter (NWK Binar pH, K-21, Landsberg, Germany) after blending 10 g of finely homogenized sample with 90 mL of double-distilled water. To estimate the cooking loss, raw meat samples were packed into Cryovac Cook-In Bags after weighing, and cooked in a water bath at 100°C for 30 min. Samples were cooled at room temperature for 1 h and reweighed. Cooking loss was calculated as the weight difference between the initial raw and final cooked samples. Water-holding capacity (WHC) was determined following the method of Kristensen and Purslow et al. (2001). Five grams of meat sample was heated to 70°C in a water bath for 30 min. Samples were then cooled with ice and subsequently centrifuged at 4°C at 1,000 × g for 10 min. WHC (%) was calculated as the ratio of weight loss of the sample during centrifugation, to that of the original liquid. Drip loss (%) was measured for 3 cm × 3 cm cuts of breast meat, which were weighed, hung in a zipper bag, and stored at 4°C. After storage, moisture on the surface of the meat slices was carefully removed and weighed at d 1, 3, 5, and 7 after the sample was taken. The initial and final weight of each sample was used to calculate drip loss. The liver, spleen, bursa of Fabricius, breast meat, abdominal fat, and gizzard were removed and weighed. Organ weights, breast meat, and abdominal fat were expressed as a percentage of live BW. Statistical Analysis All data were statistically analyzed using the GLM procedure in SAS program (SAS Institute Inc., Cary, NC). Polynomial contrasts were used to determine linear, quadratic, cubic, and quartic effects of increasing Spirulina levels on all measurements. Cage was used as an experimental unit for growth performance, nutrient digestibility, and excreta noxious gas. The individual bird was used as the experimental unit for blood oxidant enzyme, cecal microflora, and meat quality measurements. Alpha was set at 0.05. RESULTS Growth Performance Dietary Spirulina supplementation linearly increased for BWG during d 8 to 21, 22 to 35, and overall d 1 to 35, as the inclusion rate increased from 0 to 1.0% (P < 0.05) (Table 3). Increasing dietary supplementation of Spirulina had a positive linear effect on the FCR during d 8 to 21 and overall (P < 0.05). The EPEI was linearly increased associated with the inclusion of graded levels of Spirulina in the diets. No treatment effects were observed on FI and mortality throughout all the phases of feeding. Table 3. The effect of dietary Spirulina supplementation on growth performance in broilers.1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM6 Linear Quadratic Cubic Quartic d 1 to 7  BWG2, g 80 82 80 83 84 1.30 0.0736 0.4412 0.5805 0.2245  FI3, g 105 105 104 104 103 1.66 0.3858 0.9489 0.8201 0.6992  FCR4 1.309 1.285 1.292 1.254 1.232 0.028 0.0426 0.6853 0.8665 0.5565 d 8 to 21  BWG, g 637 645 646 658 665 8.10 0.0117 0.7731 0.9536 0.6035  FI, g 997 999 1,003 1,013 994 9.29 0.8367 0.3088 0.2805 0.6226  FCR 1.568 1.549 1.556 1.539 1.496 0.020 0.0199 0.3344 0.4054 0.7668 d 22 to 35  BWG, g 1,011 1,033 1,035 1,046 1,052 10.70 0.0077 0.5776 0.6815 0.6497  FI, g 1,753 1,784 1,773 1,789 1,792 14.31 0.0759 0.6158 0.5242 0.3930  FCR 1.735 1.729 1.715 1.711 1.706 0.019 0.2216 0.8641 0.9064 0.8453 Overall  BWG, g 1,729 1,760 1,762 1,787 1,801 14.58 0.0003 0.8313 0.6945 0.4344  FI, g 2,856 2,888 2,880 2,906 2,889 18.37 0.1507 0.3573 0.9714 0.3424  FCR 1.653 1.642 1.636 1.626 1.605 0.012 0.0044 0.5902 0.6570 0.9959  Mortality 5.2 5.3 5.0 4.4 4.2 0.59 0.7974 0.7811 0.6878 0.5317  EPEI5 284.2 291.4 292.6 301.7 308.0 4.08 0.0004 0.6911 0.7997 0.4758 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM6 Linear Quadratic Cubic Quartic d 1 to 7  BWG2, g 80 82 80 83 84 1.30 0.0736 0.4412 0.5805 0.2245  FI3, g 105 105 104 104 103 1.66 0.3858 0.9489 0.8201 0.6992  FCR4 1.309 1.285 1.292 1.254 1.232 0.028 0.0426 0.6853 0.8665 0.5565 d 8 to 21  BWG, g 637 645 646 658 665 8.10 0.0117 0.7731 0.9536 0.6035  FI, g 997 999 1,003 1,013 994 9.29 0.8367 0.3088 0.2805 0.6226  FCR 1.568 1.549 1.556 1.539 1.496 0.020 0.0199 0.3344 0.4054 0.7668 d 22 to 35  BWG, g 1,011 1,033 1,035 1,046 1,052 10.70 0.0077 0.5776 0.6815 0.6497  FI, g 1,753 1,784 1,773 1,789 1,792 14.31 0.0759 0.6158 0.5242 0.3930  FCR 1.735 1.729 1.715 1.711 1.706 0.019 0.2216 0.8641 0.9064 0.8453 Overall  BWG, g 1,729 1,760 1,762 1,787 1,801 14.58 0.0003 0.8313 0.6945 0.4344  FI, g 2,856 2,888 2,880 2,906 2,889 18.37 0.1507 0.3573 0.9714 0.3424  FCR 1.653 1.642 1.636 1.626 1.605 0.012 0.0044 0.5902 0.6570 0.9959  Mortality 5.2 5.3 5.0 4.4 4.2 0.59 0.7974 0.7811 0.6878 0.5317  EPEI5 284.2 291.4 292.6 301.7 308.0 4.08 0.0004 0.6911 0.7997 0.4758 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Body weight gain. 3Feed intake. 4Feed conversion ratio. 5European production efficiency index. 6Standard error of means. View Large Table 3. The effect of dietary Spirulina supplementation on growth performance in broilers.1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM6 Linear Quadratic Cubic Quartic d 1 to 7  BWG2, g 80 82 80 83 84 1.30 0.0736 0.4412 0.5805 0.2245  FI3, g 105 105 104 104 103 1.66 0.3858 0.9489 0.8201 0.6992  FCR4 1.309 1.285 1.292 1.254 1.232 0.028 0.0426 0.6853 0.8665 0.5565 d 8 to 21  BWG, g 637 645 646 658 665 8.10 0.0117 0.7731 0.9536 0.6035  FI, g 997 999 1,003 1,013 994 9.29 0.8367 0.3088 0.2805 0.6226  FCR 1.568 1.549 1.556 1.539 1.496 0.020 0.0199 0.3344 0.4054 0.7668 d 22 to 35  BWG, g 1,011 1,033 1,035 1,046 1,052 10.70 0.0077 0.5776 0.6815 0.6497  FI, g 1,753 1,784 1,773 1,789 1,792 14.31 0.0759 0.6158 0.5242 0.3930  FCR 1.735 1.729 1.715 1.711 1.706 0.019 0.2216 0.8641 0.9064 0.8453 Overall  BWG, g 1,729 1,760 1,762 1,787 1,801 14.58 0.0003 0.8313 0.6945 0.4344  FI, g 2,856 2,888 2,880 2,906 2,889 18.37 0.1507 0.3573 0.9714 0.3424  FCR 1.653 1.642 1.636 1.626 1.605 0.012 0.0044 0.5902 0.6570 0.9959  Mortality 5.2 5.3 5.0 4.4 4.2 0.59 0.7974 0.7811 0.6878 0.5317  EPEI5 284.2 291.4 292.6 301.7 308.0 4.08 0.0004 0.6911 0.7997 0.4758 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM6 Linear Quadratic Cubic Quartic d 1 to 7  BWG2, g 80 82 80 83 84 1.30 0.0736 0.4412 0.5805 0.2245  FI3, g 105 105 104 104 103 1.66 0.3858 0.9489 0.8201 0.6992  FCR4 1.309 1.285 1.292 1.254 1.232 0.028 0.0426 0.6853 0.8665 0.5565 d 8 to 21  BWG, g 637 645 646 658 665 8.10 0.0117 0.7731 0.9536 0.6035  FI, g 997 999 1,003 1,013 994 9.29 0.8367 0.3088 0.2805 0.6226  FCR 1.568 1.549 1.556 1.539 1.496 0.020 0.0199 0.3344 0.4054 0.7668 d 22 to 35  BWG, g 1,011 1,033 1,035 1,046 1,052 10.70 0.0077 0.5776 0.6815 0.6497  FI, g 1,753 1,784 1,773 1,789 1,792 14.31 0.0759 0.6158 0.5242 0.3930  FCR 1.735 1.729 1.715 1.711 1.706 0.019 0.2216 0.8641 0.9064 0.8453 Overall  BWG, g 1,729 1,760 1,762 1,787 1,801 14.58 0.0003 0.8313 0.6945 0.4344  FI, g 2,856 2,888 2,880 2,906 2,889 18.37 0.1507 0.3573 0.9714 0.3424  FCR 1.653 1.642 1.636 1.626 1.605 0.012 0.0044 0.5902 0.6570 0.9959  Mortality 5.2 5.3 5.0 4.4 4.2 0.59 0.7974 0.7811 0.6878 0.5317  EPEI5 284.2 291.4 292.6 301.7 308.0 4.08 0.0004 0.6911 0.7997 0.4758 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Body weight gain. 3Feed intake. 4Feed conversion ratio. 5European production efficiency index. 6Standard error of means. View Large Antioxidant Enzyme Activity Antioxidant enzyme activity of Spirulina was evaluated by analyzing serum SOD and GPx, and a linear increase in these enzymes was observed with increasing dietary levels of Spirulina (P < 0.0016 and P < 0.0001) (Table 4). Table 4. The effect of dietary Spirulina supplementation on blood SOD and GPx in broilers (d 35).1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM4 Linear Quadratic Cubic Quartic SOD2, U/mL 4.4 5.5 5.8 5.9 6.2 0.31 0.0016 0.1332 0.3568 0.9541 GPx3, mU/mL 38.6 44.7 43.2 46.0 45.6 0.76 <0.0001 0.0096 0.0794 0.0109 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM4 Linear Quadratic Cubic Quartic SOD2, U/mL 4.4 5.5 5.8 5.9 6.2 0.31 0.0016 0.1332 0.3568 0.9541 GPx3, mU/mL 38.6 44.7 43.2 46.0 45.6 0.76 <0.0001 0.0096 0.0794 0.0109 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Superoxide dismutase. 3Glutathione peroxidase. 4Standard error of means. View Large Table 4. The effect of dietary Spirulina supplementation on blood SOD and GPx in broilers (d 35).1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM4 Linear Quadratic Cubic Quartic SOD2, U/mL 4.4 5.5 5.8 5.9 6.2 0.31 0.0016 0.1332 0.3568 0.9541 GPx3, mU/mL 38.6 44.7 43.2 46.0 45.6 0.76 <0.0001 0.0096 0.0794 0.0109 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM4 Linear Quadratic Cubic Quartic SOD2, U/mL 4.4 5.5 5.8 5.9 6.2 0.31 0.0016 0.1332 0.3568 0.9541 GPx3, mU/mL 38.6 44.7 43.2 46.0 45.6 0.76 <0.0001 0.0096 0.0794 0.0109 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Superoxide dismutase. 3Glutathione peroxidase. 4Standard error of means. View Large Apparent Total Tract Digestibility The apparent total tract digestibility of dry matter and nitrogen linearly increased in broiler chickens fed diets supplemented with 0 to 1.0% Spirulina (P < 0.05) (Table 5). Table 5. The effect of dietary Spirulina supplementation on apparent total tract nutrient digestibility in broilers.1 Spirulina, % P-value Items, % 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Dry matter 67.9 68.5 69.7 70.1 71.1 0.68 0.0325 0.7084 0.7160 0.9944 Nitrogen 66.1 66.6 67.0 67.9 68.7 0.78 0.0157 0.6724 0.9796 0.8838 Spirulina, % P-value Items, % 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Dry matter 67.9 68.5 69.7 70.1 71.1 0.68 0.0325 0.7084 0.7160 0.9944 Nitrogen 66.1 66.6 67.0 67.9 68.7 0.78 0.0157 0.6724 0.9796 0.8838 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Standard error of means. View Large Table 5. The effect of dietary Spirulina supplementation on apparent total tract nutrient digestibility in broilers.1 Spirulina, % P-value Items, % 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Dry matter 67.9 68.5 69.7 70.1 71.1 0.68 0.0325 0.7084 0.7160 0.9944 Nitrogen 66.1 66.6 67.0 67.9 68.7 0.78 0.0157 0.6724 0.9796 0.8838 Spirulina, % P-value Items, % 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Dry matter 67.9 68.5 69.7 70.1 71.1 0.68 0.0325 0.7084 0.7160 0.9944 Nitrogen 66.1 66.6 67.0 67.9 68.7 0.78 0.0157 0.6724 0.9796 0.8838 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Standard error of means. View Large Cecal Microbial Count There was no significant difference in coliform bacteria counts of broiler chickens fed with different levels of Spirulina. However, Lactobacillus counts were significantly increased linearly as dietary Spirulina supplementation increased (P < 0.05) (Table 6). Table 6. The effect of dietary Spirulina supplementation on cecal microflora in broilers (d 35).1 Spirulina, % P-value Items, log10 cfu/g 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Lactobacillus 7.39 7.45 7.50 7.63 7.78 0.10 0.0092 0.4988 0.9625 0.8822 Coliforms 5.05 5.03 5.02 5.02 4.99 0.03 0.1179 0.9852 0.7172 0.8297 Spirulina, % P-value Items, log10 cfu/g 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Lactobacillus 7.39 7.45 7.50 7.63 7.78 0.10 0.0092 0.4988 0.9625 0.8822 Coliforms 5.05 5.03 5.02 5.02 4.99 0.03 0.1179 0.9852 0.7172 0.8297 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Standard error of means. View Large Table 6. The effect of dietary Spirulina supplementation on cecal microflora in broilers (d 35).1 Spirulina, % P-value Items, log10 cfu/g 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Lactobacillus 7.39 7.45 7.50 7.63 7.78 0.10 0.0092 0.4988 0.9625 0.8822 Coliforms 5.05 5.03 5.02 5.02 4.99 0.03 0.1179 0.9852 0.7172 0.8297 Spirulina, % P-value Items, log10 cfu/g 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Lactobacillus 7.39 7.45 7.50 7.63 7.78 0.10 0.0092 0.4988 0.9625 0.8822 Coliforms 5.05 5.03 5.02 5.02 4.99 0.03 0.1179 0.9852 0.7172 0.8297 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Standard error of means. View Large Excreta Noxious Gas Emissions Excreta ammonia emissions decreased as dietary Spirulina supplementation increased (linear, P < 0.05) (Table 7). However, Spirulina supplementation did not affect total mercaptan or hydrogen sulfide emissions. Table 7. The effect of dietary Spirulina supplementation on excreta gas emission in broilers.1 Spirulina, % P-value Items, ppm 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Ammonia 45.1 35.9 34.1 31.0 31.2 0.26 0.0050 0.3213 0.6759 0.3082 Hydrogen sulfide 2.8 2.0 2.1 2.0 2.2 0.16 0.8497 0.4291 0.8869 0.5810 Mercaptan 3.3 2.5 2.1 2.4 2.8 0.77 0.9200 0.9729 0.9200 0.4987 Spirulina, % P-value Items, ppm 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Ammonia 45.1 35.9 34.1 31.0 31.2 0.26 0.0050 0.3213 0.6759 0.3082 Hydrogen sulfide 2.8 2.0 2.1 2.0 2.2 0.16 0.8497 0.4291 0.8869 0.5810 Mercaptan 3.3 2.5 2.1 2.4 2.8 0.77 0.9200 0.9729 0.9200 0.4987 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Standard error of means. View Large Table 7. The effect of dietary Spirulina supplementation on excreta gas emission in broilers.1 Spirulina, % P-value Items, ppm 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Ammonia 45.1 35.9 34.1 31.0 31.2 0.26 0.0050 0.3213 0.6759 0.3082 Hydrogen sulfide 2.8 2.0 2.1 2.0 2.2 0.16 0.8497 0.4291 0.8869 0.5810 Mercaptan 3.3 2.5 2.1 2.4 2.8 0.77 0.9200 0.9729 0.9200 0.4987 Spirulina, % P-value Items, ppm 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic Ammonia 45.1 35.9 34.1 31.0 31.2 0.26 0.0050 0.3213 0.6759 0.3082 Hydrogen sulfide 2.8 2.0 2.1 2.0 2.2 0.16 0.8497 0.4291 0.8869 0.5810 Mercaptan 3.3 2.5 2.1 2.4 2.8 0.77 0.9200 0.9729 0.9200 0.4987 1Each treatment mean represents 10 replicates (16 birds/replicate). 2Standard error of means. View Large Meat Quality and Organ Weight There were no significant differences in pH, color (L*, a*, b*), cooking loss, or WHC of breast meat among the 5 treatment groups (P > 0.05) (Table 8). However, drip loss at 7 d post slaughter was significantly different among the 5 groups. Birds fed with Spirulina showed significantly lower drip loss as dietary levels of Spirulina increased (linear, P < 0.05). Relative weights of most organs (liver, spleen, gizzard, and bursa of Fabricius), breast meat, and abdominal fat were not significantly influenced by dietary supplementation of Spirulina. Table 8. The effect of dietary Spirulina supplementation on meat quality and relative organ weight in broilers (d 35).1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM3 Linear Quadratic Cubic Quartic pH value 5.74 5.89 5.85 6.15 6.15 0.17 0.0708 0.9484 0.8500 0.4222 Breast muscle color  Lightness (L*) 41.60 45.49 45.14 43.18 46.47 1.34 0.1056 0.5878 0.0955 0.7116  Redness (a*) 10.44 10.07 10.80 11.07 10.29 0.49 0.6645 0.5033 0.1920 0.8133  Yellowness (b*) 9.33 9.64 9.93 8.46 9.86 0.77 0.9631 0.8878 0.2597 0.3436 Cooking loss 30.11 29.97 29.61 28.69 28.54 1.27 0.4882 0.9540 0.9484 0.9145 WHC2, % 40.05 39.53 39.16 38.39 37.61 2.39 0.3575 0.6348 0.9257 0.9452 Drip loss, %  d 1 5.73 5.59 5.54 5.51 5.36 0.72 0.7671 0.9667 0.8441 0.9246  d 3 9.19 8.90 8.79 8.68 8.65 0.26 0.1472 0.6068 0.9082 0.9296  d 5 11.34 10.92 10.86 10.82 10.76 0.28 0.1814 0.4833 0.6633 0.9004  d 7 13.34 13.05 12.93 12.88 12.82 0.09 0.0009 0.1174 0.4892 0.9760 Relative organ weight, %  Breast muscle 18.55 19.10 19.13 19.61 19.73 0.87 0.3156 0.9020 0.9529 0.8111  Liver 2.86 2.80 3.07 2.88 2.77 1.34 0.8885 0.4932 0.7157 0.4625  Bursa of Fabricius 0.17 0.14 0.13 0.15 0.15 0.77 0.5623 0.1967 0.4832 0.5627  Abdominal fat 3.29 2.91 2.85 2.81 2.70 0.34 0.2624 0.6639 0.7225 0.9389  Spleen 0.13 0.11 0.10 0.11 0.11 0.01 0.3614 0.1943 0.6437 0.8215  Gizzard 1.30 1.18 1.14 1.16 1.13 0.07 0.1420 0.3963 0.5769 0.8689 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM3 Linear Quadratic Cubic Quartic pH value 5.74 5.89 5.85 6.15 6.15 0.17 0.0708 0.9484 0.8500 0.4222 Breast muscle color  Lightness (L*) 41.60 45.49 45.14 43.18 46.47 1.34 0.1056 0.5878 0.0955 0.7116  Redness (a*) 10.44 10.07 10.80 11.07 10.29 0.49 0.6645 0.5033 0.1920 0.8133  Yellowness (b*) 9.33 9.64 9.93 8.46 9.86 0.77 0.9631 0.8878 0.2597 0.3436 Cooking loss 30.11 29.97 29.61 28.69 28.54 1.27 0.4882 0.9540 0.9484 0.9145 WHC2, % 40.05 39.53 39.16 38.39 37.61 2.39 0.3575 0.6348 0.9257 0.9452 Drip loss, %  d 1 5.73 5.59 5.54 5.51 5.36 0.72 0.7671 0.9667 0.8441 0.9246  d 3 9.19 8.90 8.79 8.68 8.65 0.26 0.1472 0.6068 0.9082 0.9296  d 5 11.34 10.92 10.86 10.82 10.76 0.28 0.1814 0.4833 0.6633 0.9004  d 7 13.34 13.05 12.93 12.88 12.82 0.09 0.0009 0.1174 0.4892 0.9760 Relative organ weight, %  Breast muscle 18.55 19.10 19.13 19.61 19.73 0.87 0.3156 0.9020 0.9529 0.8111  Liver 2.86 2.80 3.07 2.88 2.77 1.34 0.8885 0.4932 0.7157 0.4625  Bursa of Fabricius 0.17 0.14 0.13 0.15 0.15 0.77 0.5623 0.1967 0.4832 0.5627  Abdominal fat 3.29 2.91 2.85 2.81 2.70 0.34 0.2624 0.6639 0.7225 0.9389  Spleen 0.13 0.11 0.10 0.11 0.11 0.01 0.3614 0.1943 0.6437 0.8215  Gizzard 1.30 1.18 1.14 1.16 1.13 0.07 0.1420 0.3963 0.5769 0.8689 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Water holding capacity. 3Standard error of means. View Large Table 8. The effect of dietary Spirulina supplementation on meat quality and relative organ weight in broilers (d 35).1 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM3 Linear Quadratic Cubic Quartic pH value 5.74 5.89 5.85 6.15 6.15 0.17 0.0708 0.9484 0.8500 0.4222 Breast muscle color  Lightness (L*) 41.60 45.49 45.14 43.18 46.47 1.34 0.1056 0.5878 0.0955 0.7116  Redness (a*) 10.44 10.07 10.80 11.07 10.29 0.49 0.6645 0.5033 0.1920 0.8133  Yellowness (b*) 9.33 9.64 9.93 8.46 9.86 0.77 0.9631 0.8878 0.2597 0.3436 Cooking loss 30.11 29.97 29.61 28.69 28.54 1.27 0.4882 0.9540 0.9484 0.9145 WHC2, % 40.05 39.53 39.16 38.39 37.61 2.39 0.3575 0.6348 0.9257 0.9452 Drip loss, %  d 1 5.73 5.59 5.54 5.51 5.36 0.72 0.7671 0.9667 0.8441 0.9246  d 3 9.19 8.90 8.79 8.68 8.65 0.26 0.1472 0.6068 0.9082 0.9296  d 5 11.34 10.92 10.86 10.82 10.76 0.28 0.1814 0.4833 0.6633 0.9004  d 7 13.34 13.05 12.93 12.88 12.82 0.09 0.0009 0.1174 0.4892 0.9760 Relative organ weight, %  Breast muscle 18.55 19.10 19.13 19.61 19.73 0.87 0.3156 0.9020 0.9529 0.8111  Liver 2.86 2.80 3.07 2.88 2.77 1.34 0.8885 0.4932 0.7157 0.4625  Bursa of Fabricius 0.17 0.14 0.13 0.15 0.15 0.77 0.5623 0.1967 0.4832 0.5627  Abdominal fat 3.29 2.91 2.85 2.81 2.70 0.34 0.2624 0.6639 0.7225 0.9389  Spleen 0.13 0.11 0.10 0.11 0.11 0.01 0.3614 0.1943 0.6437 0.8215  Gizzard 1.30 1.18 1.14 1.16 1.13 0.07 0.1420 0.3963 0.5769 0.8689 Spirulina, % P-value Items 0 0.25 0.5 0.75 1.0 SEM3 Linear Quadratic Cubic Quartic pH value 5.74 5.89 5.85 6.15 6.15 0.17 0.0708 0.9484 0.8500 0.4222 Breast muscle color  Lightness (L*) 41.60 45.49 45.14 43.18 46.47 1.34 0.1056 0.5878 0.0955 0.7116  Redness (a*) 10.44 10.07 10.80 11.07 10.29 0.49 0.6645 0.5033 0.1920 0.8133  Yellowness (b*) 9.33 9.64 9.93 8.46 9.86 0.77 0.9631 0.8878 0.2597 0.3436 Cooking loss 30.11 29.97 29.61 28.69 28.54 1.27 0.4882 0.9540 0.9484 0.9145 WHC2, % 40.05 39.53 39.16 38.39 37.61 2.39 0.3575 0.6348 0.9257 0.9452 Drip loss, %  d 1 5.73 5.59 5.54 5.51 5.36 0.72 0.7671 0.9667 0.8441 0.9246  d 3 9.19 8.90 8.79 8.68 8.65 0.26 0.1472 0.6068 0.9082 0.9296  d 5 11.34 10.92 10.86 10.82 10.76 0.28 0.1814 0.4833 0.6633 0.9004  d 7 13.34 13.05 12.93 12.88 12.82 0.09 0.0009 0.1174 0.4892 0.9760 Relative organ weight, %  Breast muscle 18.55 19.10 19.13 19.61 19.73 0.87 0.3156 0.9020 0.9529 0.8111  Liver 2.86 2.80 3.07 2.88 2.77 1.34 0.8885 0.4932 0.7157 0.4625  Bursa of Fabricius 0.17 0.14 0.13 0.15 0.15 0.77 0.5623 0.1967 0.4832 0.5627  Abdominal fat 3.29 2.91 2.85 2.81 2.70 0.34 0.2624 0.6639 0.7225 0.9389  Spleen 0.13 0.11 0.10 0.11 0.11 0.01 0.3614 0.1943 0.6437 0.8215  Gizzard 1.30 1.18 1.14 1.16 1.13 0.07 0.1420 0.3963 0.5769 0.8689 1Each treatment mean represents 20 replicates (2 birds/replicate). 2Water holding capacity. 3Standard error of means. View Large DISCUSSION This study found that broiler chickens fed diets supplemented with Spirulina increased growth performance. The mechanism of action of Spirulina has not been clearly established, but previous studies have reported that dietary supplementation with Spirulina has positive effects on growth performance in poultry. Saxena et al. (1983) reported that White Leghorn chicks fed experimental diets containing 111 g/kg and 166 g/kg Spirulina had greater weight gains at 6 wks when Spirulina replaced groundnut cake. Venkataraman et al. (1994) reported that supplementation of 140 and 170 g/kg Spirulina, with no additional vitamins/minerals, could replace groundnut cake and fishmeal with no adverse effects on broiler performance. Raju et al. (2005) concluded that dietary inclusion of Spirulina at 0.05% can partially alleviate adverse effects of 300 ppm aflatoxin on growth rate and lymphoid organ weight of broiler chickens. It has also been reported that the amino acid pattern of Spirulina microalgae could be superior to the other vegetable feeds (e.g., soybean meal), and that they have a high amino acid digestibility (Alvarenga et al., 2011; Evans et al., 2015). In addition, Spirulina contains physiologically active substances such as carotenoid pigments, phycocyanin, polyunsaturated fatty acid, vitamins, macro- and micro-mineral elements, and many other chemical compounds (Becker, 2007; Eriksen, 2008; Maoka, 2011). These compounds confirm potential antimicrobial, antioxidant, and anti-inflammatory biological properties, or act as immune enhancers (Rathore et al., 2004; Rasool et al., 2009; Uyisenga et al., 2010; Langers et al., 2012; Abdel-Daim et al., 2013; Shokri et al., 2014). Therefore, the chemical composition and physiological functions of Spirulina seem to be involved in metabolism systems related to growth performance, and are likely to be the main cause of improvement of BWG, FCR, and EPEI in broiler chickens. GPx and SOD are generally thought to act as enzymatic free radical scavengers in cells (Abdel-Wahhab and Aly, 2005). In this study, GPx and SOD linearly increase in broiler chickens fed with Spirulina. Previous studies indicated that Spirulina contains antioxidants such as β-carotene, tocopherol, selenium, polypeptide pigment, or phenolic acids, some of which might contribute to antioxidant action together or with other various micronutrients (El-Desoky et al., 2013). Specifically, Spirulina is a rich source of phycocyanin, an antioxidant biliprotein pigment, which is related to other potent antioxidants (Khan et al., 2005). There is almost no information on antioxidant properties related to Spirulina in poultry, but there is some evidence of antioxidant activity from in vitro and several rat studies. Estrada et al. (2001) suggested that protean extracts of Spirulina had scavenging effects against hydroxyl radicals, with phycocyanin as the main component responsible for the antioxidant activity. Additionally, β-carotene and other carotenoids protect cells from oxidative stress by quenching singlet oxygen damage through a variety of mechanisms (Tinkler et al., 1994). Another probable cause is that increased levels of blood SOD and GPx confirmed in Spirulina groups may be associated with phenolic compounds. Many phenolic compounds including salicylic, trans-cinnamic, synaptic, chlorogenic, quinic, and caffeic acids present in Spirulina may also be responsible for its antioxidant activity, individually or synergistically (Miranda et al., 1998). Wu et al. (2005) suggested that Spirulina extract has stronger antioxidant capabilities than Chlorella, which is probably due to higher content of phenolic compounds (23.87 vs. 15.25 mg tannic acid equivalent/g of algae aqueous extract) and antioxidant capacity (ABTS assay: 19.74 vs. 4.60 μmol of Trolox equivalent/g of microalgae). Therefore, increased serum SOD and GPx concentrations in this study were likely due to chemical compounds such as phycocyanin, β-carotene, and phenolics in Spirulina, all relating to the antioxidant activities. In this study, the apparent total tract digestibilities of dry matter and nitrogen linearly increased in broilers fed with Spirulina diets, indicating that higher digestibility could be achieved with higher concentrations of Spirulina. The digestibility of Spirulina is not well documented, and the available studies on assimilation by poultry have not provided conclusive results. However, Mabeau and Fleurence (1993) confirmed that marine microalgae showed a high rate of protein degradation proteolytic enzymes such as pepsin, pancreatin, and pronase. Evans et al. (2015) reported that young broilers (21-d-old) had higher apparent ileal digestibility of glutamic acid, proline, glycine, alanine, methionine, leucine, and lysine when fed 6 to 21% Spirulina supplemented diets compared with broilers fed control diets. Furbeyre et al. (2017) reported that the total tract digestibility in pigs receiving 1% Spirulina and 1% Chlorella was greater for gross energy and tended to be greater for dry matter, organic matter, and neutral detergent fiber compared with control pigs. They also found that villus height at the jejunum was greater in pigs fed with Spirulina and Chlorella compared with control pigs. Microalgae are generally regarded as a viable protein source, with essential amino acid (EAA) composition meeting the Food and Agriculture Organization requirements, and are often on par with other protein sources, such as soybean and egg (Bleakley and Hayes, 2017). Increased digestibilities of dry matter and nitrogen in this study may be related to the high-quality protein containing balanced EAA. Therefore, the better digestibility of protein observed in Spirulina supplemented diets may be a result of better absorption, which enhanced growth in broiler chickens. The cecum plays an important role in preventing colonization of pathogens, detoxifying harmful substances, recycling nitrogen, microbial synthesis of vitamins, degradation of some carbohydrates, and absorbing additional nutrients (Coates et al., 1968; Clench and Mathias, 1995; Jorgensen et al., 1996). A previous study in broiler chickens also concluded that intestinal microbial-including cecum is highly associated with the production performance of broiler chickens (Jeong and Kim, 2014). Therefore, cecal microbial populations were investigated to evaluate Spirulina supplementation in broiler chickens. This study indicated that broiler chickens fed a Spirulina supplemented diet led to higher cecal Lactobacillus concentration, but there was no difference in the number of coliform bacteria. Some studies suggest that microalgae have potential antibacterial, antiviral, and antifungal activities. In vitro, de Mule et al. (1996) observed that methanolic and aqueous extracts of Spirulina inhibited the growth of Candida albicans by 17.6%, whereas Lactococcus lactis was promoted by the extract, with growth increasing from 7.5 to 14.7%. Kaushik and Chauhan (2008) demonstrated that the extracts of Spirulina have shown antibacterial effects by inhibiting the growth of harmful microorganisms, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumonia. The addition of dry Spirulina at 10 mg/mL into de Man, Rogosa, and Sharpe medium-promoted growth of Lactobacillus acidophilus by 186%, suggesting prebiotic potential of the microalgae (Bhowmik et al., 2009). Rania and Hala (2008) also suggested that Spirulina extract had antibacterial activities against E. coli because of the presence of alkaloids and lipopolysaccharides. In an in vivo study, feeding Chlorella microalgae resulted in increased Lactobacillus diversity in the crop or cecum or both of laying hens (Janczyk et al., 2009; Kang et al., 2013). To date, relatively few studies have investigated the antimicrobial activity of microalgae, including Spirulina and their extracts in poultry. We hypothesized that Spirulina supplementation would maintain the beneficial microbial population, and subsequently explain the improved digestibility and growth performance in this study. In regard to excreta noxious gas emission, the inclusion of Spirulina linearly reduced the excreta ammonia gas content as dietary levels of Spirulina increased. Excreta noxious gas emission of animals is associated with intestinal microflora, particularly harmful intestinal bacteria populations (Ferket et al., 2002). A previous study also suggested that lower excreta noxious gas content occurs when the microflora in the gastrointestinal tract of broiler chickens is manipulated (Jeong and Kim, 2014). Several other studies have also suggested that excreta noxious gas content is associated with nutrient digestibility (Yan et al., 2011; Jeong and Kim, 2014), because the increased digestibility may allow less substrate for microbial fermentation in the large intestine, which consequently decreases excreta noxious gas content. In our study, the Spirulina supplemented diet led to a better balanced microflora in the cecum and higher nutrient digestibility than that by control diet. Therefore, we suggest that the reason for reduction in excreta ammonia gas content may be the result of increased nitrogen digestibility and Lactobacillus populations in broiler chicken ceca. There is currently a lack of useful information regarding Spirulina in poultry, and previously reported functions of Spirulina in other species cannot be directly compared. However, findings from the current study support previous research (Yan et al., 2012) showing that dietary Chlorella supplementation decreased excreta ammonia gas emission in growing pigs. Venkataraman et al. (1994) showed that color pigmentation of skin, breast, and thigh muscles was deeper in broilers when substituting groundnut protein or fish meal with Spirulina up to 170 or 140 g/kg. Toyomizu et al. (2001) found that including Spirulina in broiler feeds influenced both the yellowness and redness of broiler meat. They reported that the increase of yellowness with dietary Spirulina content may be reflected in the common yellow pigment related to the accumulation of zeaxanthin within the meat. However, unlike previous reports that Spirulina has an effect on meat color, this study showed no differences in breast meat color of broiler chickens fed with Spirulina. Additionally, there were no differences in pH, cooking loss, WHC, or organ weights of broiler chickens fed with Spirulina. Drip loss causes nutrients and moisture to escape, and negatively affects chewiness of meat. Spirulina-supplemented diets showed a significant reduction of drip loss after 7 d of storage. However, reasons for this decrease remain unknown. Lu et al. (2014) suggested that dietary addition of antioxidants was effective in improving growth, moderately restored whole body antioxidant capability, and reduced drip loss. Asghar et al. (1989) and Attia et al. (2016) indicated that antioxidants preserve the functionality of membranes, thus improving their role as semipermeable barriers against exudative loss. According to Cheah et al. (1995), dietary antioxidant supplementation also prevents excessive drip loss from pale, soft, exudative muscle from stress-susceptible pigs. This action seems to result from decreased membrane phospholipase activity through higher antioxidant content of the tissue membranes, because drip loss occurs when phospholipids in the intracellular membrane are oxidized and the membrane becomes weak. In this study, decreased drip loss due to the inclusion of dietary Spirulina is, presumably, related to the delayed oxidation of the cell membrane. Improvement was probably due to the positive effect of bioactive compounds, including antioxidants of Spirulina, on the integrity of muscle fibers; thus, enhancing their capability to retain water (Dal Bosco et al., 2014). The findings of our study support previous research, which determined that Spirulina could affect meat quality by decreasing the drip loss of Japanese quail meats (Cheong et al., 2016). Further studies are needed to determine the mechanisms involved in the association between Spirulina and drip loss of meat. In conclusion, supplementation with Spirulina improved BWG, FCR, and EPEI; increased antioxidant enzyme activity, dry matter and nitrogen digestibilities; increased cecal Lactobacillus populations; and decreased excreta ammonia gas emission in broiler chickens. For breast meat quality, Spirulina decreased drip loss after 7 d of storage. Therefore, Spirulina microalgae at 1.0% diet might provide a good alternative to improve broiler chicken production. Acknowledgements This research was financially supported by the Ministry of Trade, Industry, and Energy (MOTIE), Korea, under the “Regional Specialized Industry Development Program”(reference number R0005540) supervised by the Korea Institute for Advancement of Technology (KIAT). REFERENCES Abdel-Daim M. M. , Abuzead S. M. , Halawa S. M. . 2013 . Protective role of Spirulina platensis against acute deltamethrin-induced toxicity in rats . PLoS One . 8 : e72991 . Google Scholar CrossRef Search ADS PubMed Abdel-Wahhab M. A. , Aly S. E. . 2005 . Antioxidant property of Nigella sativa (black cumin) and Syzygium aromaticum (clove) in rats during aflatoxicosis . J. Appl. Toxicol. 25 : 218 – 223 . Google Scholar CrossRef Search ADS PubMed Al-Batshan H. A. , Al-Mufarrej S. I. , Al-Homaidan A. A. , Qureshi M. A. . 2001 . Enhancement of chicken macrophage phagocytic function and nitrite production by dietary Spirulina platensis . Immunopharmacol. Immunotoxicol. 23 : 281 – 289 . Google Scholar CrossRef Search ADS PubMed Alvarenga R. R. , Rodrigues P. B. , Cantarelli V. , Zangeronimo M. G. , Da Silva Junior J. W. , Da Silva L. R. , Dos Santos L. M. , Pereira L. J. . 2011 . Energy values and chemical composition of Spirulina (Spirulina platensis) evaluated with broilers . Braz. J. Anim. Sci. 40 : 992 – 996 . AOAC . 2000 . Official Method of Analysis . 17th ed . Assoc. Off. Anal. Chem. , Washington, DC . PubMed PubMed Asghar A. , Lin C. F. , Gray J. I. , Buckley D. J. , Booren A. M. , Crackel R. L. , Flegal C. J. . 1989 . Influence of oxidised oil and antioxidant supplementation on membrane bound lipid stability in broiler meat . Br. Poult. Sci. 30 : 815 – 823 . Google Scholar CrossRef Search ADS PubMed Attia Y. A. , Al-Harthi M. A. , Korish M. M. , Shiboob M. M. . 2016 . Evaluation of the broiler meat quality in the retail market: Effects of type and source of carcasses . Rev. Mex. Cienc. Pecu. 7 : 321 – 339 . Becker E. W. 2007 . Micro-algae as a source of protein . Biotechnol. Adv. 25 : 207 – 210 . Google Scholar CrossRef Search ADS PubMed Bhavisha R. , Parula P. . 2010 . Spirulina: potential clinical therapeutic application . J. Pharm. Res. 3 : 1726 – 1732 . Bhowmik D. , Dubey J. , Mehra S. . 2009 . Probiotic efficiency of Spirulina platensis: stimulating growth of lactic acid bacteria . Am. Eurasian J. Agric. Environ. Sci. 6 : 546 – 549 . Bleakley S. , Hayes M. . 2017 . Algal proteins: extraction, application, and challenges concerning production . Foods 26 : 6 . pii: E33 . Cheah K. S. , Cheah A. M. , Krausgrill D. I. . 1995 . Effect of dietary supplementation of vitamin E on pig meat quality . Meat Sci. 39 : 255 – 264 . Google Scholar CrossRef Search ADS PubMed Cheong D. S. W. , Kasim A. , Sazili A. Q. , Omar H. , Teoh J. Y. . 2016 . Effect of supplementing Spirulina on live performance, carcass composition and meat quality of Japanese quail . Walailak J. Sci. Tech. 13 : 77 – 84 . Clench M. H. , Mathias J. R. . 1995 . The avian cecum: a review . Wilson Bull . 107 : 93 – 121 . Coates M. E. , Ford J. E. , Harrison G. F. . 1968 . Intestinal synthesis of vitamins of the B complex in chicks . Br. J. Nutr. 22 : 493 – 498 . Google Scholar CrossRef Search ADS PubMed Dal Bosco A. , Gerencsér Z. , Szendrő Z. , Mugnai C. , Cullere M. , Kovács M. , Ruggeri S. , Mattioli S. , Castellini C. , Dalle Zotte A. . 2014 . Effect of dietary supplementation of Spirulina (Arthrospira platensis) and Thyme (Thymus vulgaris) on rabbit meat appearance, oxidative stability and fatty acid profile during retail display . Meat Sci. 96 : 114 – 119 . Google Scholar CrossRef Search ADS PubMed de Mule´ M. C. Z. , de Caire G. Z. , de Cano M. S. . 1996 . Bioactive substances from Spirulina platensis (Cyanobacteria) . Phyton . 58 : 93 – 96 . El-Desoky G. E. , Bashandy S. A. , Alhazza I. M. , Al-Othman Z. A. , Aboul-Soud M. A. , Yusuf K. . 2013 . Improvement of mercuric chloride-induced testis injuries and sperm quality deteriorations by Spirulina platensis in rats . PLoS One . 8 : e59177 . Google Scholar CrossRef Search ADS PubMed Eriksen N. T. 2008 . Production of phycocyanin—a pigment with applications in biology, biotechnology, foods and medicine . Appl. Microbiol. Biotechnol. 80 : 1 – 14 . Google Scholar CrossRef Search ADS PubMed Evans A. M. , Smith D. L. , Moritz J. S. . 2015 . Effects of algae incorporation into broiler starter diet formulations on nutrient digestibility and 3 to 21 d bird performance . J. Appl. Poult. Res. 24 : 206 – 214 . Google Scholar CrossRef Search ADS Estrada J. P. , Bescós P. B. , Del Fresno A. V. . 2001 . Antioxidant activity of different fractions of Spirulina platensis protean extract . Il Farmaco . 56 : 497 – 500 . Google Scholar CrossRef Search ADS PubMed Ferket P. R. , van Heugten E. , van Kempen T. A. T. G. , Angel R. . 2002 . Nutritional strategies to reduce environmental emissions from nonruminants . J. Anim. Sci. 80 : E168 – E182 . Google Scholar CrossRef Search ADS Furbeyre H. , van Milgen J. , Mener T. , Gloaguen M. , Labussiere E. . 2017 . Effects of dietary supplementation with freshwater microalgae on growth performance, nutrient digestibility and gut health in weaned piglets . Animal . 11 : 183 – 192 . Google Scholar CrossRef Search ADS PubMed Janczyk P. , Halle B. , Souffrant W. B. . 2009 . Microbial community composition of the crop and ceca contents of laying hens fed diets supplemented with Chlorella vulgaris . Poult. Sci. 88 : 2324 – 2332 . Google Scholar CrossRef Search ADS PubMed Jeong J. S. , Kim I. H. . 2014 . Effect of Bacillus subtilis C-3102 spores as a probiotic feed supplement on growth performance, noxious gas emission, and intestinal microflora in broilers . Poult. Sci. 93 : 3097 – 3103 . Google Scholar CrossRef Search ADS PubMed Jorgensen H. , Zhao X. Q. , Knudsen K. E. , Eggum B. O. . 1996 . The influence of dietary fibre source and level on the development of the gastrointestinal tract, digestibility and energy metabolism in broiler chickens . Br. J. Nutr. 75 : 379 – 395 . Google Scholar CrossRef Search ADS PubMed Joventino I. P. , Alves H. G. , Neves L. C. , Pinheiro-Joventino F. , Leal L. K. , Neves S. A. , Ferreira F. V. , Brito G. A. , Viana G. B. . 2012 . The microalga Spirulina platensis presents anti-inflammatory action as well as hypoglycemic and hypolipidemic properties in diabetic rats . J. Complement. Integr. Med. 9 : 17 . Google Scholar CrossRef Search ADS Kang H. K. , Salim H. M. , Akter N. , Kim D. W. , Kim J. H. , Bang H. T. , Kim M. J. , Na J. C. , Hwangbo J. , Choi H. C. , Suh O. S. . 2013 . Effect of various forms of dietary Chlorella supplementation on growth performance, immune characteristics, and intestinal microflora population of broiler chickens . J. Appl. Poult. Res. 22 : 100 – 108 . Google Scholar CrossRef Search ADS Kaushik P. , Chauhan A. . 2008 . In vitro antibacterial activity of laboratory grown culture of Spirulina platensis . Indian J. Microbiol. 48 : 348 – 352 . Google Scholar CrossRef Search ADS PubMed Khan Z. , Bhadouria P. , Bisen P. S. . 2005 . Nutritional and therapeutic potential of Spirulina . Curr. Pharm. Biotechnol. 6 : 373 – 379 . Google Scholar CrossRef Search ADS PubMed Korean Feeding Standards for Poultry . 2012 . National Institute of Animal Science , RDA , Suwon, Republic of Korea . Kristensen L. , Purslow P. P. . 2001 . The effect of ageing on the water-holding capacity of pork: role of cytoskeletal proteins . Meat Sci. 58 : 17 – 23 . Google Scholar CrossRef Search ADS PubMed Kulshreshtha A. , Jarouliya U. , Bhadauriya P. , Prasad G. B. K. S. , Bisen P. S. . 2008 . Spirulina in health care management . Curr. Pharm. Biotechnol. 9 : 400 – 405 . Google Scholar CrossRef Search ADS PubMed Langers I. , Renoux V. M. , Thiry M. , Delvenne P. , Jacobs N. . 2012 . Natural killer cells: role in local tumor growth and metastasis . Biologics . 6 : 73 – 82 . Google Scholar PubMed Lu T. , Harper A. F. , Zhao J. , Dalloul R. A. . 2014 . Effects of a dietary antioxidant blend and vitamin E on growth performance, oxidative status, and meat quality in broiler chickens fed a diet high in oxidants . Poult. Sci. 93 : 1649 – 1657 . Google Scholar CrossRef Search ADS PubMed Mabeau S. , Fleurence J. . 1993 . Seaweed in food products: biochemical and nutritional aspects . Trends Food Sci. Technol. 4 : 103 – 107 . Google Scholar CrossRef Search ADS Meineri G. , Ingravalle F. , Radice E. , Aragno M. , Peiretti P. G. , 2009 : Effects of high fat diets and Spirulina platensis supplementation in New Zealand white rabbits . J. Anim. Vet. Adv. 8 : 2735 – 2744 . Maoka T. 2011 . Carotenoids in marine animals . Marine Drugs . 9 : 278 – 293 . Google Scholar CrossRef Search ADS PubMed Miranda M. S. , Cintra R. G. , Barros S. B. M. , Filho J. M. . 1998 . Antioxidant activity of the microalga Spirulina maxima . Braz. J. Med. Biol. Res. 31 : 1075 – 1079 . Google Scholar CrossRef Search ADS PubMed National research council (NRC) . 1994 . Nutrient Requirements of Poultry . 9th rev. ed . National Academy Press , Washington DC . Raju M. V. L. N. , Rao S. V. , Radhika K. , Chawak M. M. . 2005 . Dietary supplementation of Spirulina and its effects on broiler chicken exposed to aflatoxicosis . Indian J. Poult. Sci. 40 : 36 – 40 . Rania M. A. , Hala M. T. . 2008 . Antibacterial and antifungal activity of cyanobacteria and green microalgae. Evaluation of medium components by placket-burman design for antimicrobial activity of Spirulina platensis . Global J. Biotechnol. Biochem. 3 : 22 – 31 . Rasool M. K. , Sabina E. P. , Nithya P. , Lavanya K. . 2009 . Suppressive effect of Spirulina fusiformis in relation to lysosomal acid hydrolases, lipid peroxidation, antioxidant status, and inflammatory mediator TNF-alpha on experimental gouty arthritis in mice . Orient. Pharm. Exp. Med. 9 : 164 – 173 . Google Scholar CrossRef Search ADS Rathore N. K. , Singh S. , Singh V. . 2004 . Spirulina as immuno-enhancer and biomodulator . J. Immunol. Immunopathol. 6 : 1 – 10 . Saxena P. N. , Ahmad M. R. , Shyam R. , Amla D. V. . 1983 . Cultivation of Spirulina in sewage for poultry feed . Experientia 39 : 1077 – 1083 . Google Scholar CrossRef Search ADS Shields R. J. , Lupatsch I. . 2012 . Algae for aquaculture and animal feeds . J. Anim. Sci. 21 : 23 – 37 . Shokri H. , Khosravi A. , Taghavi M. . 2014 . Efficacy of Spirulina platensis on immune functions in cancer mice with systemic candidiasis . J. Mycol. Res. 1 : 7 – 13 . Sujatha T. , Narahari D. . 2011 . Effect of designer diets on egg yolk composition of ‘White Leghorn’ hens . J. Food Sci. Technol. 48 : 494 – 497 . Google Scholar CrossRef Search ADS PubMed Tinkler J. H. , Bohm F. , Schalch W. , Truscott T. G. . 1994 . Dietary carotenoids protect human cells from damage . J. Photochem. Photobiol. 26 : 283 – 285 . Google Scholar CrossRef Search ADS Toyomizu M. , Sato K. , Taroda H. , Kato T. , Akiba Y. . 2001 . Effects of dietary Spirulina on meat colour in muscle of broiler chickens . Br. Poult. Sci. 42 : 197 – 202 . Google Scholar CrossRef Search ADS PubMed Uyisenga J. P. , Nzayino P. , Seneza R. , Hishamunda L. , Uwantege K. , Gasana N. , Emmanuel S. B. . 2010 . In vitro study of antibacterial and antifungal activity of Spirulina platensis . Int. J. Ecol. Dev. 16 : 80 – 88 . Venkataraman L. V. , Somasekaran T. , Becker E. W. . 1994 . Replacement value of blue-green alga (Spirulina platensis) for fishmeal and a vitamin-mineral premix for broiler chicks . Br. Poult. Sci. 35 : 373 – 381 . Google Scholar CrossRef Search ADS PubMed Wu L. C. , Ho J. A. A. , Shieh M. C. , Lu I. W. . 2005 . Antioxidant and antiproliferative activities of Spirulina and Chlorella water extracts . J. Agric. Food Chem. 53 : 4207 – 4212 . Google Scholar CrossRef Search ADS PubMed Yan L. , Meng Q. W. , Kim I. H. . 2011 . The effect of an herb extract mixture on growth performance, nutrient digestibility, blood characteristics and fecal noxious gas content in growing pigs . Livestock Sci. 141 : 143 – 147 . Google Scholar CrossRef Search ADS Yan L. , Lim S. U. , Kim I. H. . 2012 . Effect of fermented chlorella supplementation on growth performance, nutrient digestibility, blood characteristics, fecal microbial and fecal noxious gas content in growing pigs . Asian Australas. J. Anim. Sci 25 : 1742 – 1747 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the term of oxford University Press, standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Poultry ScienceOxford University Press

Published: Jun 22, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off