Dietary supplementation with DHA-rich microalgae improves performance, serum composition, carcass trait, antioxidant status, and fatty acid profile of broilers

Dietary supplementation with DHA-rich microalgae improves performance, serum composition, carcass... ABSTRACT This experiment was conducted with 126 as-hatched male Arbor Acres chicks (1-d-old, weighing 45.3 ± 0.72 g) to determine the effects of microalgae [MA, containing 29% docosahexaenoic acid (DHA)] on performance, serum composition, carcass trait, antioxidant status, and fatty acid deposition of birds. The birds were allocated randomly to 1 of 3 treatments with 7 replicate pens per treatment (6 birds per pen). The dietary treatments included a control diet [corn-soybean basal diet supplemented with 3% soybean oil (SO), CON], 1% MA diet (basal diet supplemented with 1% MA and 2% SO, 1MA), and 2% MA diet (basal diet supplemented with 2% MA and 1% SO, 2MA). All birds were raised in wire-floored cages. The trial consists of a starter phase from d 1 to 21 and a grower phase from d 22 to 42. Compared with CON, birds supplemented with MA (1MA or 2MA) had greater (P < 0.05) average daily gain, liver percentage (liver weight/body weight), and serum glucose, as well as lower (P < 0.05) feed conversation ratio, abdominal fat percentage (abdominal fat weight/body weight), and total serum cholesterol. Moreover, due to the high concentration of DHA in MA, birds fed MA showed increased (P < 0.05) concentration of eicosapentaenoic acid, DHA, superoxide dismutase, and total antioxidant capacity, as well as decreased (P < 0.05) n-6/n-3 polyunsaturated fatty acid ratio, polyunsaturated fatty acid/saturated fatty acid ratio, and malondialdehyde in the breast and thigh muscle compared with CON. In conclusion, dietary supplementation with 1% or 2% DHA-rich microalgae had positive effects on performance, serum composition, carcass trait, antioxidant status, and fatty acid deposition in birds. INTRODUCTION Dietary intakes of high level long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), particularly eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), are suboptimal in humans’ diets (Givens and Gibbs, 2008; Adkins and Kelley, 2010). However, LC n-3 PUFA and DHA are crucial for decreasing risk of cancer, atherosclerosis, cardiovascular disease, coronary heart disease, and other related diseases in humans (Singh et al., 1997; Marckmann and Gronbæk, 1999; Zhang et al., 2010) because they can help to reduce concentrations of serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (Bang and Dyerberg, 1972; Abudabos et al., 2013). Researchers have reported that DHA is rich in marine fish and some marine plants, especially some strains of microalgae (MA) et al., 2016). One efficient approach to increase intakes of DHA is via the enrichment of basic foods for humans (Alasalvar et al., 2002; Arterburn et al., 2006; Schmitz and Ecker, 2008). Since poultry meat has become one of the most consumed meats with an annual average per capita consumption of 22 kg in Europe (Eurostat, 2008) and 39 kg in the United States (USDA, 2010), scientists have focused on manipulating fatty acid composition of poultry meat through altering fatty acid composition of broiler diets, with the goal of increasing DHA consumption in human diets (Lopez-Ferrer et al., 2001a; Rymer and Givens, 2005; Givens and Gibbs, 2008). Supplementation of animal diets with lipids and oils rich in n-3 PUFA is regarded as an efficient way to improve the concentration of DHA and EPA in animal tissues (Lopez-Ferrer et al., 2001b). Usually, the enrichment of LC n-3 PUFA in poultry meat is realized by adding fish oil to the broiler diet (Rymer and Givens, 2005). However, use of fish oil may reduce oxidative stability of poultry meat (O’Keefe et al., 1995; Bou et al., 2001) because high concentration of PUFA in fish oil is easily to produced off-flavors (Meynier et al., 1999), and off-odors (Kahraman et al., 2004; Wood et al., 2008). An alternative approach is to feed birds with diets containing marine algae to produce high-oxidative stability chicken meat (Mooney et al., 1998; Guschina and Harwood, 2006), because marine algae (the primary producers of DHA) have high content of DHA and some antioxidants, including beta-carotenoids, vitamin A, and vitamin E (Barclay et al., 1994). Low level of Algal biomass (1% to 5%) has been demonstrated to be effective in improving DHA concentration of pig meat (Sardi et al., 2006), eggs (Cheng et al., 2005; Cachaldora et al., 2008), and poultry meat (Mooney et al., 1998; Kralik et al., 2004). Moreover, MA from Schizochytrium sp. are rich in DHA and vitamins (e.g., beta-carotene and vitamin E), and often used as one of the best candidates to produce sustainable and affordable DHA (Hakim, 2013). However, there are few studies focusing on the effects of low level of MA from Schizochytrium sp. on both fatty acid deposition and antioxidant status. So the exact impact of adding low level of MA replacing soybean oil (SO) into broilers’ diets on broiler performance, carcass trait, and lipid profile remains to be established although it is well known that marine algae are excellent sources of DHA. The objective of the experiment reported in this paper was to investigate the effect of adding different levels of a DHA-rich MA strain replacing SO on performance, serum composition, carcass trait, antioxidant status, and deposition of LC n-3 PUFA in broilers. MATERIALS AND MMETHODS All the procedures used in this animal experiment were approved by the Institutional Animal Care and Use Committee of China Agricultural University (Beijing, China). Experimental Product A commercial source of dehydrated, whole-cell MA, All-G-Rich (Schizochytrium limacinum CCAP 4087/2), which contains 64% fat, 29% DHA, 11% crude protein, 2.04% vitamin A, and 0.07% vitamin E, was supplied by Alltech Inc. (Nicholasville, KY). The SO was obtained from Beijing Tongli Xing Department of Agricultural Science and Technology Company Limited (Beijing, China). Fatty acid composition of MA and SO used in the experiment is displayed in Table 1. Table 1. Fatty acid composition of microalgae and soybean oil used in experimental diets (% total fat). Fatty acid  MA1  SO1  C14:0 Myristic acid  5.15  0.08  C16:0 Palmitic acid  60.1  10.9  C16:1n-7 Palmitoleic acid  0.13  0.09  C17:0 Heptadecanoic acid  0.56  0.10  C18:0 Stearic acid  1.79  4.40  C18:1n-9 Oleic acid  0.03  20.1  C18:2n-6 Linoleic acid  0.04  53.5  C18:3n-6 Methyl linolenate  0.08  0.08  C18:3n-3 α-Linolenic acid  0.05  7.86  C20:0 Arachidic acid  0.29  0.00  C20:1n-9 Twitocene-enoic acid  0.12  0.03  C20:4n-6 Arachidonic acid  0.08  0.00  C20:5n-3 Eicosapentaenoic acid  0.30  0.01  C22:6n-3 Docosahexaenoic acid  28.7  0.04  Saturated fatty acids  67.9  15.5  Monounsaturated fatty acids  0.15  20.1  Polyunsaturated fatty acids  29.2  61.5  n-6 Polyunsaturated fatty acids  0.19  53.6  n-3 Polyunsaturated fatty acids  29.1  7.91  n-6/n-32  0.01  6.77  P/S2  0.43  3.97  Fatty acid  MA1  SO1  C14:0 Myristic acid  5.15  0.08  C16:0 Palmitic acid  60.1  10.9  C16:1n-7 Palmitoleic acid  0.13  0.09  C17:0 Heptadecanoic acid  0.56  0.10  C18:0 Stearic acid  1.79  4.40  C18:1n-9 Oleic acid  0.03  20.1  C18:2n-6 Linoleic acid  0.04  53.5  C18:3n-6 Methyl linolenate  0.08  0.08  C18:3n-3 α-Linolenic acid  0.05  7.86  C20:0 Arachidic acid  0.29  0.00  C20:1n-9 Twitocene-enoic acid  0.12  0.03  C20:4n-6 Arachidonic acid  0.08  0.00  C20:5n-3 Eicosapentaenoic acid  0.30  0.01  C22:6n-3 Docosahexaenoic acid  28.7  0.04  Saturated fatty acids  67.9  15.5  Monounsaturated fatty acids  0.15  20.1  Polyunsaturated fatty acids  29.2  61.5  n-6 Polyunsaturated fatty acids  0.19  53.6  n-3 Polyunsaturated fatty acids  29.1  7.91  n-6/n-32  0.01  6.77  P/S2  0.43  3.97  1MA: microalgae; SO: soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Table 1. Fatty acid composition of microalgae and soybean oil used in experimental diets (% total fat). Fatty acid  MA1  SO1  C14:0 Myristic acid  5.15  0.08  C16:0 Palmitic acid  60.1  10.9  C16:1n-7 Palmitoleic acid  0.13  0.09  C17:0 Heptadecanoic acid  0.56  0.10  C18:0 Stearic acid  1.79  4.40  C18:1n-9 Oleic acid  0.03  20.1  C18:2n-6 Linoleic acid  0.04  53.5  C18:3n-6 Methyl linolenate  0.08  0.08  C18:3n-3 α-Linolenic acid  0.05  7.86  C20:0 Arachidic acid  0.29  0.00  C20:1n-9 Twitocene-enoic acid  0.12  0.03  C20:4n-6 Arachidonic acid  0.08  0.00  C20:5n-3 Eicosapentaenoic acid  0.30  0.01  C22:6n-3 Docosahexaenoic acid  28.7  0.04  Saturated fatty acids  67.9  15.5  Monounsaturated fatty acids  0.15  20.1  Polyunsaturated fatty acids  29.2  61.5  n-6 Polyunsaturated fatty acids  0.19  53.6  n-3 Polyunsaturated fatty acids  29.1  7.91  n-6/n-32  0.01  6.77  P/S2  0.43  3.97  Fatty acid  MA1  SO1  C14:0 Myristic acid  5.15  0.08  C16:0 Palmitic acid  60.1  10.9  C16:1n-7 Palmitoleic acid  0.13  0.09  C17:0 Heptadecanoic acid  0.56  0.10  C18:0 Stearic acid  1.79  4.40  C18:1n-9 Oleic acid  0.03  20.1  C18:2n-6 Linoleic acid  0.04  53.5  C18:3n-6 Methyl linolenate  0.08  0.08  C18:3n-3 α-Linolenic acid  0.05  7.86  C20:0 Arachidic acid  0.29  0.00  C20:1n-9 Twitocene-enoic acid  0.12  0.03  C20:4n-6 Arachidonic acid  0.08  0.00  C20:5n-3 Eicosapentaenoic acid  0.30  0.01  C22:6n-3 Docosahexaenoic acid  28.7  0.04  Saturated fatty acids  67.9  15.5  Monounsaturated fatty acids  0.15  20.1  Polyunsaturated fatty acids  29.2  61.5  n-6 Polyunsaturated fatty acids  0.19  53.6  n-3 Polyunsaturated fatty acids  29.1  7.91  n-6/n-32  0.01  6.77  P/S2  0.43  3.97  1MA: microalgae; SO: soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Experimental Animals and Management As-hatched Arbor Acres chicks (n = 126; 1 day of age, weighing 45.3 ± 0.72 g) were purchased from Arbor Acres Poultry Breeding Company (Beijing, China). All birds were raised in wire-floored cages in an environmentally controlled room with continuous light (10 to 20 lux) and were permitted ad libitum access to feed and water. Room temperature was maintained at 33°C for the first 3 d, then the temperature was reduced gradually by 3°C per week until reaching 24°C which was maintained until the end of the 42-d experiment. The lighting regimen and ventilation were monitored continuously from d 1 to 42. All birds were inoculated with Newcastle disease vaccine on d 7 and 28 and with inactivated infectious bursa disease vaccine on d 14 and 21. The trial was conducted in 2 phases consisting of a starter phase from d 1 to 21 (phase 1) and a grower phase from d 22 to 42 (phase 2). Experimental Design and Diets Broilers were allotted randomly to 1 of 3 dietary treatments (Table 2). The dietary treatments included a control diet [corn-soybean basal diet supplemented with 3% soybean oil (SO), CON], 1% MA diet (basal diet supplemented with 1% MA and 2% SO, 1MA), 2% MA diet (basal diet supplemented with 2% MA and 1% SO, 2MA). There were 7 replicate pens per treatment with 6 birds per pen. All essential nutrients contained in the basal diet in phase 1 and phase 2 met the nutrients requirements suggested by the NRC (1994). All diets were fed in mash form. Table 2. Composition and nutrient levels of experimental diets (%, as-fed basis). Item  Phase 1 (d 1 to 21)  Phase 2 (d 22 to 42)    CON1  1MA1  2MA1  CON  1MA  2MA  Corn  58.70  58.70  58.70  65.40  65.40  65.40  Soybean meal  30.11  30.11  30.11  22.69  22.69  22.69  Corn gluten meal  4.00  4.00  4.00  5.10  5.10  5.10  Soybean oil  3.00  2.00  1.00  3.00  2.00  1.00  Microalgae  -  1.00  2.00  -  1.00  2.00  Dicalcium phosphate  1.70  1.70  1.70  1.25  1.25  1.25  Limestone  1.39  1.39  1.39  1.44  1.44  1.44  Salt  0.30  0.30  0.30  0.30  0.30  0.30  L-lysine HCl, 78%  0.12  0.12  0.12  0.21  0.21  0.21  DL-Methionine, 98%  0.15  0.15  0.15  0.05  0.05  0.05  L-Threonine, 98%  0.03  0.03  0.03  0.06  0.06  0.06  Vitamin-mineral Premix2  0.50  0.50  0.50  0.50  0.50  0.50  Nutrient composition              Metabolic Energy, MJ/kg3  12.76  12.69  12.61  13.18  13.12  13.04  Crude Protein4  20.70  20.93  20.46  20.10  19.88  19.86  Calcium3  1.00  1.00  1.00  0.90  0.90  0.90  Available Phosphorus3  0.45  0.45  0.45  0.35  0.35  0.35  SID lysine3  1.10  1.10  1.10  1.00  1.00  1.00  SID methionine3  0.50  0.50  0.50  0.38  0.38  0.38  SID threonine3  0.80  0.80  0.80  0.74  0.74  0.74  SID tryptophan3  0.27  0.27  0.27  0.23  0.23  0.23  Item  Phase 1 (d 1 to 21)  Phase 2 (d 22 to 42)    CON1  1MA1  2MA1  CON  1MA  2MA  Corn  58.70  58.70  58.70  65.40  65.40  65.40  Soybean meal  30.11  30.11  30.11  22.69  22.69  22.69  Corn gluten meal  4.00  4.00  4.00  5.10  5.10  5.10  Soybean oil  3.00  2.00  1.00  3.00  2.00  1.00  Microalgae  -  1.00  2.00  -  1.00  2.00  Dicalcium phosphate  1.70  1.70  1.70  1.25  1.25  1.25  Limestone  1.39  1.39  1.39  1.44  1.44  1.44  Salt  0.30  0.30  0.30  0.30  0.30  0.30  L-lysine HCl, 78%  0.12  0.12  0.12  0.21  0.21  0.21  DL-Methionine, 98%  0.15  0.15  0.15  0.05  0.05  0.05  L-Threonine, 98%  0.03  0.03  0.03  0.06  0.06  0.06  Vitamin-mineral Premix2  0.50  0.50  0.50  0.50  0.50  0.50  Nutrient composition              Metabolic Energy, MJ/kg3  12.76  12.69  12.61  13.18  13.12  13.04  Crude Protein4  20.70  20.93  20.46  20.10  19.88  19.86  Calcium3  1.00  1.00  1.00  0.90  0.90  0.90  Available Phosphorus3  0.45  0.45  0.45  0.35  0.35  0.35  SID lysine3  1.10  1.10  1.10  1.00  1.00  1.00  SID methionine3  0.50  0.50  0.50  0.38  0.38  0.38  SID threonine3  0.80  0.80  0.80  0.74  0.74  0.74  SID tryptophan3  0.27  0.27  0.27  0.23  0.23  0.23  1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). 2Premix supplied per kg diet: vitamin A, 11,000 IU; vitamin D, 3,025 IU; vitamin E, 22 mg; vitamin K3, 2.2 mg; thiamine, 1.65 mg; riboflavin, 6.6 mg; pyridoxine, 3.3 mg; cobalamin, 17.6 μg; nicotinic acid, 22 mg; pantothenic acid, 13.2 mg; folic acid, 0.33 mg; biotin, 88 μg; choline chloride, 500 mg; iron, 48 mg; zinc, 96.6 mg; manganese, 101.76 mg; copper, 10 mg; selenium, 0.05 mg; iodine, 0.96 mg; cobalt, 0.3 mg. 3Calculated value; SID means standardized ileal digestible. 4Analysed value. View Large Table 2. Composition and nutrient levels of experimental diets (%, as-fed basis). Item  Phase 1 (d 1 to 21)  Phase 2 (d 22 to 42)    CON1  1MA1  2MA1  CON  1MA  2MA  Corn  58.70  58.70  58.70  65.40  65.40  65.40  Soybean meal  30.11  30.11  30.11  22.69  22.69  22.69  Corn gluten meal  4.00  4.00  4.00  5.10  5.10  5.10  Soybean oil  3.00  2.00  1.00  3.00  2.00  1.00  Microalgae  -  1.00  2.00  -  1.00  2.00  Dicalcium phosphate  1.70  1.70  1.70  1.25  1.25  1.25  Limestone  1.39  1.39  1.39  1.44  1.44  1.44  Salt  0.30  0.30  0.30  0.30  0.30  0.30  L-lysine HCl, 78%  0.12  0.12  0.12  0.21  0.21  0.21  DL-Methionine, 98%  0.15  0.15  0.15  0.05  0.05  0.05  L-Threonine, 98%  0.03  0.03  0.03  0.06  0.06  0.06  Vitamin-mineral Premix2  0.50  0.50  0.50  0.50  0.50  0.50  Nutrient composition              Metabolic Energy, MJ/kg3  12.76  12.69  12.61  13.18  13.12  13.04  Crude Protein4  20.70  20.93  20.46  20.10  19.88  19.86  Calcium3  1.00  1.00  1.00  0.90  0.90  0.90  Available Phosphorus3  0.45  0.45  0.45  0.35  0.35  0.35  SID lysine3  1.10  1.10  1.10  1.00  1.00  1.00  SID methionine3  0.50  0.50  0.50  0.38  0.38  0.38  SID threonine3  0.80  0.80  0.80  0.74  0.74  0.74  SID tryptophan3  0.27  0.27  0.27  0.23  0.23  0.23  Item  Phase 1 (d 1 to 21)  Phase 2 (d 22 to 42)    CON1  1MA1  2MA1  CON  1MA  2MA  Corn  58.70  58.70  58.70  65.40  65.40  65.40  Soybean meal  30.11  30.11  30.11  22.69  22.69  22.69  Corn gluten meal  4.00  4.00  4.00  5.10  5.10  5.10  Soybean oil  3.00  2.00  1.00  3.00  2.00  1.00  Microalgae  -  1.00  2.00  -  1.00  2.00  Dicalcium phosphate  1.70  1.70  1.70  1.25  1.25  1.25  Limestone  1.39  1.39  1.39  1.44  1.44  1.44  Salt  0.30  0.30  0.30  0.30  0.30  0.30  L-lysine HCl, 78%  0.12  0.12  0.12  0.21  0.21  0.21  DL-Methionine, 98%  0.15  0.15  0.15  0.05  0.05  0.05  L-Threonine, 98%  0.03  0.03  0.03  0.06  0.06  0.06  Vitamin-mineral Premix2  0.50  0.50  0.50  0.50  0.50  0.50  Nutrient composition              Metabolic Energy, MJ/kg3  12.76  12.69  12.61  13.18  13.12  13.04  Crude Protein4  20.70  20.93  20.46  20.10  19.88  19.86  Calcium3  1.00  1.00  1.00  0.90  0.90  0.90  Available Phosphorus3  0.45  0.45  0.45  0.35  0.35  0.35  SID lysine3  1.10  1.10  1.10  1.00  1.00  1.00  SID methionine3  0.50  0.50  0.50  0.38  0.38  0.38  SID threonine3  0.80  0.80  0.80  0.74  0.74  0.74  SID tryptophan3  0.27  0.27  0.27  0.23  0.23  0.23  1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). 2Premix supplied per kg diet: vitamin A, 11,000 IU; vitamin D, 3,025 IU; vitamin E, 22 mg; vitamin K3, 2.2 mg; thiamine, 1.65 mg; riboflavin, 6.6 mg; pyridoxine, 3.3 mg; cobalamin, 17.6 μg; nicotinic acid, 22 mg; pantothenic acid, 13.2 mg; folic acid, 0.33 mg; biotin, 88 μg; choline chloride, 500 mg; iron, 48 mg; zinc, 96.6 mg; manganese, 101.76 mg; copper, 10 mg; selenium, 0.05 mg; iodine, 0.96 mg; cobalt, 0.3 mg. 3Calculated value; SID means standardized ileal digestible. 4Analysed value. View Large Sampling and Processing Diets were ground to pass through a 1-mm sieve before analysis. Diets were analyzed for concentrations of dry matter (DM; Method 934.01) and crude protein (CP; Method 990.03) according to the procedures of the Association of Official Analytical Chemists (AOAC, 2005), and gross energy was determined by an automatic isoperibolic oxygen bomb calorimeter (Parr 1281, Automatic Energy Analyzer; Moline, IL). On d 21 and 42, broilers were fasted for 12 h and the birds and feeders were then weighed to determine average daily gain (ADG), average daily feed intake (ADFI), and feed conversation ratio (FCR). One bird (closest to the average body weight for each pen) was euthanized for blood samples (n = 7). Blood (5 mL) was collected by cardiac puncture into a 10-mL anticoagulant-free Vacutainer tube (Greiner Bio-One GmbH, Kremsmunster, Austria) and then centrifuged at 3,000 × g for 10 min at 4°C to obtain serum. The serum samples were stored at −80°C until analysis. Measurement of Serum Indices Concentrations of glucose, albumin, globulin, albumin/globulin ratio, total protein (TP), urea nitrogen (UN), triglyceride, TC, LDL-C, high-density lipoprotein cholesterol (HDL-C) in serum samples were analyzed by an automatic biochemical analyzer (RA-1000, Bayer Corp., Tarrytown, NY) using colorimetric methods, following the instructions of the manufacturer of the corresponding reagent kit (Zhongsheng Biochemical Co., Ltd., Beijing, China). Determination of serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) levels were conducted by spectrophotometric methods using a spectrophotometer (Leng Guang SFZ1606017568, Shanghai, China) following the instructions of the kit's manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Determination of Carcass Traits The birds (n = 7, one bird closed to the average body weight from each pen) that were euthanized on d 42 were also used to determine carcass traits. The breast and thigh muscle located on the left side were removed and weighed. The percentage of the breast and thigh muscle weight relative to slaughter weight was determined. Meat color, including lightness (L*), redness (a*), and yellowness (b*) values, was measured from 3 orientations (middle, medial, and lateral) using a Chromameter (CR-410, Konica Minota, Tokyo, Japan). The pH values at 45 min and 24 h postmortem were also measured at 3 locations using a glass penetration pH electrode (pH-star, Matthaus, Germany). Drip loss for 24 h postmortem was measured using the plastic bag method as described previously (Straadt et al., 2007). On d 42, the organs of these broilers, including heart, liver, spleen, pancreas, abdominal fat, and fabricius, were collected and weighed to determine the organ percentages (organ percentage = organ weight/terminal body weight × 100%). Determination of Fatty Acid Composition At the end of d 42, milled (1-mm screen) feed (in duplicate) or homogenized skinless breast and thigh meat tissue (7 samples of each tissue from each diet) were defrosted and samples of feed (10 g), breast and thigh meat (ca. 20 g) were lyophilized for 60 h using a freeze dryer. Fatty acid profiles of the lipid sources were determined by gas chromatography (6890 series, Agilent Technologies, Wilmington, DE) according to the procedures of Sukhija and Palmquist (1988) with slight modifications. Lipid samples were converted to fatty acid methyl esters using methanolic HCl. Undecanoic acid (C11:0) was used as the internal standard. Aliquots of 1 liter were injected into a capillary column (60 m × 250 m × 250 nm, DB-23, Agilent) with cyanopropyl methyl silicone as the stationary phase. Column oven temperature was programmed with a 1:20 split. Injector and detector temperatures were maintained at 260 and 270°C, respectively. Nitrogen was the carrier gas at a flow rate of 2 mL/min. Fatty acid concentrations were then calculated in terms of milligrams of fatty acid per 100 g of feed or tissue (fresh weight). Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), n-6, n-3, and polyunsaturated fatty acids/saturated fatty acids ratio (P/S) were calculated using the following formulae:   \begin{equation*} {\rm{SFA}} = {\rm{C}}14:0 + {\rm{C}}16:0 + {\rm{C}}17:0 + {\rm{C}}18:0 + {\rm{C}}20:0 \end{equation*}   \begin{equation*} {\rm{MUFA}} = {\rm{C}}16:1{\rm{n}} - 7 + {\rm{C}}18:1{\rm{n}} - 9 \end{equation*}   \begin{eqnarray*} &&{\rm{PUFA}} = {\rm{C}}18:2{\rm{n}} - 6 + {\rm{C}}18:3{\rm{n}} - 6 + {\rm{C}}20\\ &&:4{\rm{n}} - 6 + {\rm{C}}18:3{\rm{n}} - 3 + {\rm{C}}20:5{\rm{n}} - 3 + {\rm{C}}22:6{\rm{n}} - 3 \end{eqnarray*}   \begin{equation*} {\rm{n}} - 6 = {\rm{C}}18:2{\rm{n}} - 6 + {\rm{C}}18:3{\rm{n}} - 6 + {\rm{C}}20:4{\rm{n}} - 6 \end{equation*}   \begin{equation*} {\rm{n}} - 3 = {\rm{C}}18:3{\rm{n}} - 3 + {\rm{C}}20:5{\rm{n}} - 3 + {\rm{C}}22:6{\rm{n}} - 3 \end{equation*}   \begin{equation*} {\rm{P}}/{\rm{S}} = {\rm{PUFA}}/{\rm{SFA}} \end{equation*} Statistical Analyses Data were subjected to ANOVA using the GLM procedure of SAS (SAS Institute, 1996). Pen was the experimental unit. Differences among treatments were separated by Duncan's multiple range test. Results were expressed as least squares means and SEM. Significance was designated at P ≤ 0.05, while a tendency for significance was designated at 0.05 < P ≤ 0.10. RESULTS Fatty Acid of Diets Fatty acid content of diets is shown in Table 3. The main fatty acids present in diets were C16:0, C18:0, C18:1n-9, C18:2n-6, C20:0. As MA increased, concentrations of DHA and n-3 PUFA had an increase of 2% to 3% per 1% increase of MA, while the n-6/n-3 PUFA ratio was decreased by a large margin. Table 3. Concentrations of fatty acids of diets in the experimental diets (% of total fat). Fatty acid  Phase 1  Phase 2    CON1  1MA1  2MA1  CON  1MA  2MA  C14:0 Myristic acid  0.09  0.06  1.15  0.08  0.07  0.62  C16:0 Palmitic acid  17.5  18.9  23.7  16.7  19.9  24.6  C16:1n-7 Palmitoleic acid  0.09  0.04  0.05  0.03  0.04  0.05  C17:0 Heptadecanoic acid  0.13  0.14  0.19  0.13  0.16  0.22  C18:0 Stearic acid  4.38  3.24  2.80  4.11  3.26  2.38  C18:1n-9 Oleic acid  26.3  21.5  19.2  24.2  20.4  17.3  C18:2n-6 Linoleic acid  42.2  46.1  41.0  46.0  46.2  42.1  C18:3n-6 Methyl linolenate  0.00  0.00  0.00  0.00  0.00  0.00  C18:3n-3 α-Linolenic acid  0.12  0.05  0.08  0.10  0.08  0.09  C20:0 Arachidic acid  3.18  3.91  3.09  3.33  3.57  2.30  C20:1n-9 Twitocene-enoic acid  0.53  0.41  0.38  0.51  0.43  0.40  C20:4n-6 Arachidonic acid  0.00  0.00  0.00  0.00  0.00  0.00  C20:5n-3 Eicosapentaenoic acid  0.03  0.00  0.05  0.05  0.03  0.07  C22:6n-3 Docosahexaenoic acid  0.28  2.58  5.48  0.27  2.43  6.04  Saturated fatty acids  25.2  26.2  31.0  24.4  26.9  30.1  Monounsaturated fatty acids  26.8  22.0  19.6  24.7  20.8  17.7  Polyunsaturated fatty acids  42.6  48.8  46.6  46.4  48.7  48.3  n-6 Polyunsaturated fatty acids  42.2  46.1  41.0  46.0  46.2  42.1  n-3 Polyunsaturated fatty acids  0.43  2.63  5.61  0.42  2.54  6.20  n-6/n-32  98.1  17.5  7.30  109  18.2  6.78  P/S2  1.69  1.86  1.50  1.90  1.81  1.60  Fatty acid  Phase 1  Phase 2    CON1  1MA1  2MA1  CON  1MA  2MA  C14:0 Myristic acid  0.09  0.06  1.15  0.08  0.07  0.62  C16:0 Palmitic acid  17.5  18.9  23.7  16.7  19.9  24.6  C16:1n-7 Palmitoleic acid  0.09  0.04  0.05  0.03  0.04  0.05  C17:0 Heptadecanoic acid  0.13  0.14  0.19  0.13  0.16  0.22  C18:0 Stearic acid  4.38  3.24  2.80  4.11  3.26  2.38  C18:1n-9 Oleic acid  26.3  21.5  19.2  24.2  20.4  17.3  C18:2n-6 Linoleic acid  42.2  46.1  41.0  46.0  46.2  42.1  C18:3n-6 Methyl linolenate  0.00  0.00  0.00  0.00  0.00  0.00  C18:3n-3 α-Linolenic acid  0.12  0.05  0.08  0.10  0.08  0.09  C20:0 Arachidic acid  3.18  3.91  3.09  3.33  3.57  2.30  C20:1n-9 Twitocene-enoic acid  0.53  0.41  0.38  0.51  0.43  0.40  C20:4n-6 Arachidonic acid  0.00  0.00  0.00  0.00  0.00  0.00  C20:5n-3 Eicosapentaenoic acid  0.03  0.00  0.05  0.05  0.03  0.07  C22:6n-3 Docosahexaenoic acid  0.28  2.58  5.48  0.27  2.43  6.04  Saturated fatty acids  25.2  26.2  31.0  24.4  26.9  30.1  Monounsaturated fatty acids  26.8  22.0  19.6  24.7  20.8  17.7  Polyunsaturated fatty acids  42.6  48.8  46.6  46.4  48.7  48.3  n-6 Polyunsaturated fatty acids  42.2  46.1  41.0  46.0  46.2  42.1  n-3 Polyunsaturated fatty acids  0.43  2.63  5.61  0.42  2.54  6.20  n-6/n-32  98.1  17.5  7.30  109  18.2  6.78  P/S2  1.69  1.86  1.50  1.90  1.81  1.60  1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Table 3. Concentrations of fatty acids of diets in the experimental diets (% of total fat). Fatty acid  Phase 1  Phase 2    CON1  1MA1  2MA1  CON  1MA  2MA  C14:0 Myristic acid  0.09  0.06  1.15  0.08  0.07  0.62  C16:0 Palmitic acid  17.5  18.9  23.7  16.7  19.9  24.6  C16:1n-7 Palmitoleic acid  0.09  0.04  0.05  0.03  0.04  0.05  C17:0 Heptadecanoic acid  0.13  0.14  0.19  0.13  0.16  0.22  C18:0 Stearic acid  4.38  3.24  2.80  4.11  3.26  2.38  C18:1n-9 Oleic acid  26.3  21.5  19.2  24.2  20.4  17.3  C18:2n-6 Linoleic acid  42.2  46.1  41.0  46.0  46.2  42.1  C18:3n-6 Methyl linolenate  0.00  0.00  0.00  0.00  0.00  0.00  C18:3n-3 α-Linolenic acid  0.12  0.05  0.08  0.10  0.08  0.09  C20:0 Arachidic acid  3.18  3.91  3.09  3.33  3.57  2.30  C20:1n-9 Twitocene-enoic acid  0.53  0.41  0.38  0.51  0.43  0.40  C20:4n-6 Arachidonic acid  0.00  0.00  0.00  0.00  0.00  0.00  C20:5n-3 Eicosapentaenoic acid  0.03  0.00  0.05  0.05  0.03  0.07  C22:6n-3 Docosahexaenoic acid  0.28  2.58  5.48  0.27  2.43  6.04  Saturated fatty acids  25.2  26.2  31.0  24.4  26.9  30.1  Monounsaturated fatty acids  26.8  22.0  19.6  24.7  20.8  17.7  Polyunsaturated fatty acids  42.6  48.8  46.6  46.4  48.7  48.3  n-6 Polyunsaturated fatty acids  42.2  46.1  41.0  46.0  46.2  42.1  n-3 Polyunsaturated fatty acids  0.43  2.63  5.61  0.42  2.54  6.20  n-6/n-32  98.1  17.5  7.30  109  18.2  6.78  P/S2  1.69  1.86  1.50  1.90  1.81  1.60  Fatty acid  Phase 1  Phase 2    CON1  1MA1  2MA1  CON  1MA  2MA  C14:0 Myristic acid  0.09  0.06  1.15  0.08  0.07  0.62  C16:0 Palmitic acid  17.5  18.9  23.7  16.7  19.9  24.6  C16:1n-7 Palmitoleic acid  0.09  0.04  0.05  0.03  0.04  0.05  C17:0 Heptadecanoic acid  0.13  0.14  0.19  0.13  0.16  0.22  C18:0 Stearic acid  4.38  3.24  2.80  4.11  3.26  2.38  C18:1n-9 Oleic acid  26.3  21.5  19.2  24.2  20.4  17.3  C18:2n-6 Linoleic acid  42.2  46.1  41.0  46.0  46.2  42.1  C18:3n-6 Methyl linolenate  0.00  0.00  0.00  0.00  0.00  0.00  C18:3n-3 α-Linolenic acid  0.12  0.05  0.08  0.10  0.08  0.09  C20:0 Arachidic acid  3.18  3.91  3.09  3.33  3.57  2.30  C20:1n-9 Twitocene-enoic acid  0.53  0.41  0.38  0.51  0.43  0.40  C20:4n-6 Arachidonic acid  0.00  0.00  0.00  0.00  0.00  0.00  C20:5n-3 Eicosapentaenoic acid  0.03  0.00  0.05  0.05  0.03  0.07  C22:6n-3 Docosahexaenoic acid  0.28  2.58  5.48  0.27  2.43  6.04  Saturated fatty acids  25.2  26.2  31.0  24.4  26.9  30.1  Monounsaturated fatty acids  26.8  22.0  19.6  24.7  20.8  17.7  Polyunsaturated fatty acids  42.6  48.8  46.6  46.4  48.7  48.3  n-6 Polyunsaturated fatty acids  42.2  46.1  41.0  46.0  46.2  42.1  n-3 Polyunsaturated fatty acids  0.43  2.63  5.61  0.42  2.54  6.20  n-6/n-32  98.1  17.5  7.30  109  18.2  6.78  P/S2  1.69  1.86  1.50  1.90  1.81  1.60  1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Bird Performance Performance of birds is summarized in Table 4. Compared with CON, birds supplemented with 1MA had greater (P < 0.01) body weight (BW) on d 21 and d 42, ADG in phase 1, 2 and overall (d 1 to 42), as well as higher (P < 0.01) ADFI over the 42 d experiment. Birds fed with 2MA showed higher (P < 0.01) ADG in phase 1, whereas they had lower (P < 0.01) ADFI in phase 2 and over the 42 d experiment compared with CON. Birds supplemented with MA (1MA or 2MA) had lower (P < 0.05) FCR in phase 1 and overall, while birds fed with 2MA had lower (P < 0.01) FCR in phase 2 compared with CON. Table 4. Effects of DHA-rich microalgae on performance of broilers. Item  CON1  1MA  2MA  SEM  P-value   d 0 body weight, g  44.9  46.0  45.0  0.72  0.54   d 21 body weight, g  652b  729a  707a  12.1  < 0.01   d 42 body weight, g  2,380b  2,632a  2,445b  32.5  < 0.01  d 1 to 21   Average daily gain, g/d  28.9b  32.5a  31.5a  0.57  < 0.01   Average daily feed intake, g/d  43.3  45.0  43.5  0.95  0.41   Feed conversation ratio, g/g  1.50a  1.39b  1.38b  0.03  0.02  d 22 to 42   Average daily gain, g/d  82.3b  90.7a  82.8b  1.40  < 0.01   Average daily feed intake, g/d  139a  149a  124b  3.33  < 0.01   Feed conversation ratio, g/g  1.70a  1.65a  1.50b  0.04  < 0.01  d 1 to 42   Average daily gain, g/d  55.6b  61.6a  57.1b  0.78  < 0.01   Average daily feed intake, g/d  91.3b  97.1a  83.7c  1.70  < 0.01   Feed conversation ratio, g/g  1.60a  1.52b  1.44b  0.03  < 0.01  Item  CON1  1MA  2MA  SEM  P-value   d 0 body weight, g  44.9  46.0  45.0  0.72  0.54   d 21 body weight, g  652b  729a  707a  12.1  < 0.01   d 42 body weight, g  2,380b  2,632a  2,445b  32.5  < 0.01  d 1 to 21   Average daily gain, g/d  28.9b  32.5a  31.5a  0.57  < 0.01   Average daily feed intake, g/d  43.3  45.0  43.5  0.95  0.41   Feed conversation ratio, g/g  1.50a  1.39b  1.38b  0.03  0.02  d 22 to 42   Average daily gain, g/d  82.3b  90.7a  82.8b  1.40  < 0.01   Average daily feed intake, g/d  139a  149a  124b  3.33  < 0.01   Feed conversation ratio, g/g  1.70a  1.65a  1.50b  0.04  < 0.01  d 1 to 42   Average daily gain, g/d  55.6b  61.6a  57.1b  0.78  < 0.01   Average daily feed intake, g/d  91.3b  97.1a  83.7c  1.70  < 0.01   Feed conversation ratio, g/g  1.60a  1.52b  1.44b  0.03  < 0.01  SEM means standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). View Large Table 4. Effects of DHA-rich microalgae on performance of broilers. Item  CON1  1MA  2MA  SEM  P-value   d 0 body weight, g  44.9  46.0  45.0  0.72  0.54   d 21 body weight, g  652b  729a  707a  12.1  < 0.01   d 42 body weight, g  2,380b  2,632a  2,445b  32.5  < 0.01  d 1 to 21   Average daily gain, g/d  28.9b  32.5a  31.5a  0.57  < 0.01   Average daily feed intake, g/d  43.3  45.0  43.5  0.95  0.41   Feed conversation ratio, g/g  1.50a  1.39b  1.38b  0.03  0.02  d 22 to 42   Average daily gain, g/d  82.3b  90.7a  82.8b  1.40  < 0.01   Average daily feed intake, g/d  139a  149a  124b  3.33  < 0.01   Feed conversation ratio, g/g  1.70a  1.65a  1.50b  0.04  < 0.01  d 1 to 42   Average daily gain, g/d  55.6b  61.6a  57.1b  0.78  < 0.01   Average daily feed intake, g/d  91.3b  97.1a  83.7c  1.70  < 0.01   Feed conversation ratio, g/g  1.60a  1.52b  1.44b  0.03  < 0.01  Item  CON1  1MA  2MA  SEM  P-value   d 0 body weight, g  44.9  46.0  45.0  0.72  0.54   d 21 body weight, g  652b  729a  707a  12.1  < 0.01   d 42 body weight, g  2,380b  2,632a  2,445b  32.5  < 0.01  d 1 to 21   Average daily gain, g/d  28.9b  32.5a  31.5a  0.57  < 0.01   Average daily feed intake, g/d  43.3  45.0  43.5  0.95  0.41   Feed conversation ratio, g/g  1.50a  1.39b  1.38b  0.03  0.02  d 22 to 42   Average daily gain, g/d  82.3b  90.7a  82.8b  1.40  < 0.01   Average daily feed intake, g/d  139a  149a  124b  3.33  < 0.01   Feed conversation ratio, g/g  1.70a  1.65a  1.50b  0.04  < 0.01  d 1 to 42   Average daily gain, g/d  55.6b  61.6a  57.1b  0.78  < 0.01   Average daily feed intake, g/d  91.3b  97.1a  83.7c  1.70  < 0.01   Feed conversation ratio, g/g  1.60a  1.52b  1.44b  0.03  < 0.01  SEM means standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). View Large Carcass Traits Effects of MA on carcass traits of birds are shown in Table 5. Breast muscle yield was lower (P < 0.05) in birds fed with 2MA compared with those supplemented with 1MA. Thigh muscle yield tended to be higher (P = 0.10) in birds supplemented with 1MA compared with CON. There were no negative effects of MA supplementation on thigh and breast pH value, lightness, redness, yellowness, or drip loss of birds, while MA increased (P < 0.05) liver percentage (liver weight/body weight) and decreased (P < 0.05) abdominal fat percentage (abdominal fat weight/body weight) compared with CON. Birds supplemented with 2MA had higher (P < 0.05) spleen percentage (spleen weight/body weight) compared with CON. Table 5. Effects of DHA-rich microalgae on carcass traits of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle   Breast muscle yield, %  7.90a,b  8.45a  7.28b  0.26  0.03   pH40 min  6.45  6.41  6.57  0.08  0.38   pH24 h  5.26  5.33  5.44  0.08  0.28   Lightness  38.0  37.6  37.1  0.70  0.67   Redness  3.34  3.41  2.90  0.47  0.72   Yellowness  8.94  9.00  9.16  0.31  0.89   Drip loss, %  2.14  1.91  1.76  0.53  0.83  Thigh muscle   Thigh muscle yield, %  9.44d  10.2c  9.34d  0.28  0.10   pH40 min  6.46  6.40  6.36  0.07  0.67   pH24 h  5.30  5.34  5.23  0.08  0.61   Lightness  39.1  39.2  40.2  0.88  0.64   Redness  4.40  4.68  4.76  0.35  0.75   Yellowness  10.0  10.4  10.3  0.44  0.85   Drip loss, %  0.71  0.87  1.08  0.13  0.17  Slaughter weight, kg  2.51  2.59  2.43  54.2  0.16   Heart index percentage  0.58  0.71  0.67  0.07  0.42   Liver percentage  1.98b  2.56a  2.49a  0.14  0.02   Spleen percentage  1.07b  1.21b  1.78a  0.16  0.02   Pancreas percentage  0.17  0.19  0.20  0.01  0.23   Abdominal fat percentage  0.20a  0.15b  0.14b  0.02  0.04   Fabricius percentage  0.24  0.23  0.22  0.03  0.88  Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle   Breast muscle yield, %  7.90a,b  8.45a  7.28b  0.26  0.03   pH40 min  6.45  6.41  6.57  0.08  0.38   pH24 h  5.26  5.33  5.44  0.08  0.28   Lightness  38.0  37.6  37.1  0.70  0.67   Redness  3.34  3.41  2.90  0.47  0.72   Yellowness  8.94  9.00  9.16  0.31  0.89   Drip loss, %  2.14  1.91  1.76  0.53  0.83  Thigh muscle   Thigh muscle yield, %  9.44d  10.2c  9.34d  0.28  0.10   pH40 min  6.46  6.40  6.36  0.07  0.67   pH24 h  5.30  5.34  5.23  0.08  0.61   Lightness  39.1  39.2  40.2  0.88  0.64   Redness  4.40  4.68  4.76  0.35  0.75   Yellowness  10.0  10.4  10.3  0.44  0.85   Drip loss, %  0.71  0.87  1.08  0.13  0.17  Slaughter weight, kg  2.51  2.59  2.43  54.2  0.16   Heart index percentage  0.58  0.71  0.67  0.07  0.42   Liver percentage  1.98b  2.56a  2.49a  0.14  0.02   Spleen percentage  1.07b  1.21b  1.78a  0.16  0.02   Pancreas percentage  0.17  0.19  0.20  0.01  0.23   Abdominal fat percentage  0.20a  0.15b  0.14b  0.02  0.04   Fabricius percentage  0.24  0.23  0.22  0.03  0.88  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). View Large Table 5. Effects of DHA-rich microalgae on carcass traits of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle   Breast muscle yield, %  7.90a,b  8.45a  7.28b  0.26  0.03   pH40 min  6.45  6.41  6.57  0.08  0.38   pH24 h  5.26  5.33  5.44  0.08  0.28   Lightness  38.0  37.6  37.1  0.70  0.67   Redness  3.34  3.41  2.90  0.47  0.72   Yellowness  8.94  9.00  9.16  0.31  0.89   Drip loss, %  2.14  1.91  1.76  0.53  0.83  Thigh muscle   Thigh muscle yield, %  9.44d  10.2c  9.34d  0.28  0.10   pH40 min  6.46  6.40  6.36  0.07  0.67   pH24 h  5.30  5.34  5.23  0.08  0.61   Lightness  39.1  39.2  40.2  0.88  0.64   Redness  4.40  4.68  4.76  0.35  0.75   Yellowness  10.0  10.4  10.3  0.44  0.85   Drip loss, %  0.71  0.87  1.08  0.13  0.17  Slaughter weight, kg  2.51  2.59  2.43  54.2  0.16   Heart index percentage  0.58  0.71  0.67  0.07  0.42   Liver percentage  1.98b  2.56a  2.49a  0.14  0.02   Spleen percentage  1.07b  1.21b  1.78a  0.16  0.02   Pancreas percentage  0.17  0.19  0.20  0.01  0.23   Abdominal fat percentage  0.20a  0.15b  0.14b  0.02  0.04   Fabricius percentage  0.24  0.23  0.22  0.03  0.88  Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle   Breast muscle yield, %  7.90a,b  8.45a  7.28b  0.26  0.03   pH40 min  6.45  6.41  6.57  0.08  0.38   pH24 h  5.26  5.33  5.44  0.08  0.28   Lightness  38.0  37.6  37.1  0.70  0.67   Redness  3.34  3.41  2.90  0.47  0.72   Yellowness  8.94  9.00  9.16  0.31  0.89   Drip loss, %  2.14  1.91  1.76  0.53  0.83  Thigh muscle   Thigh muscle yield, %  9.44d  10.2c  9.34d  0.28  0.10   pH40 min  6.46  6.40  6.36  0.07  0.67   pH24 h  5.30  5.34  5.23  0.08  0.61   Lightness  39.1  39.2  40.2  0.88  0.64   Redness  4.40  4.68  4.76  0.35  0.75   Yellowness  10.0  10.4  10.3  0.44  0.85   Drip loss, %  0.71  0.87  1.08  0.13  0.17  Slaughter weight, kg  2.51  2.59  2.43  54.2  0.16   Heart index percentage  0.58  0.71  0.67  0.07  0.42   Liver percentage  1.98b  2.56a  2.49a  0.14  0.02   Spleen percentage  1.07b  1.21b  1.78a  0.16  0.02   Pancreas percentage  0.17  0.19  0.20  0.01  0.23   Abdominal fat percentage  0.20a  0.15b  0.14b  0.02  0.04   Fabricius percentage  0.24  0.23  0.22  0.03  0.88  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). View Large Serological Index Effects of MA on serological index of birds are presented in Table 6. In phase 1, birds fed 2MA showed higher (P < 0.05) levels of serum glucose and albumin/globulin ratio, while birds supplemented with MA had lower (P ≤ 0.05) concentration of TC and LDL-C in serum compared with CON. In phase 2, birds supplemented with MA had higher (P < 0.01) concentration of glucose and a trend to increase (P = 0.09) TP, while birds fed with MA also showed lower (P < 0.01) concentration of serum UN and tended to decrease (P = 0.08) concentration of TC in serum compared with CON. Concentration of LDL-C in serum tended to be lower (P = 0.07) in birds supplemented with 1MA, whereas content of HDL-C in serum tended to increase (P = 0.09) in birds fed with 2MA in phase 2 compared with CON. Table 6. Effects of DHA-rich microalgae on serological index of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  d 1 to 21   Glucose, mmol/L  17.6b  18.7a,b  19.6a  0.43  0.03   Albumin, g/L  11.9  12.6  12.9  0.72  0.66   Globulin, g/L  18.7  19.9  19.9  1.24  0.84   Albumin/globulin ratio  0.63b  0.63b  0.66a  0.01  0.02   Total protein, g/L  30.6  32.5  32.8  1.95  0.99   Urea nitrogen, mmol/L  0.48  0.50  0.44  0.04  0.95   Triglycerides, mmol/L  0.99  0.43  0.76  0.14  0.22   Total cholesterol, mmol/L  4.57a  3.62b  3.42b  0.21  0.05   LDL-C, mmol/L2  0.52a  0.38b  0.43b  0.03  0.05   HDL-C, mmol/L2  1.53  1.62  1.61  0.07  0.56  d 22 to 42   Glucose, mmol/L  14.5b  18.6a  16.9a  0.63  < 0.01   Albumin, g/L  11.6  12.8  12.6  0.39  0.22   Globulin, g/L  16.2  22.0  20.8  1.75  0.14   Albumin/globulin ratio  0.73  0.60  0.61  0.05  0.23   Total protein, g/L  27.8d  34.8c  33.4c  1.95  0.09   Urea nitrogen, mmol/L  0.40a  0.23b  0.34a  0.02  < 0.01   Triglycerides, mmol/L  0.77  0.70  0.50  0.09  0.46   Total cholesterol, mmol/L  3.30c  2.73d  2.70d  0.16  0.08   LDL-C, mmol/L  0.57c  0.34d  0.42c,d  0.05  0.07   HDL-C, mmol/L  1.13d  1.25c,d  1.40c  0.09  0.09  Item  CON1  1MA1  2MA1  SEM  P-value  d 1 to 21   Glucose, mmol/L  17.6b  18.7a,b  19.6a  0.43  0.03   Albumin, g/L  11.9  12.6  12.9  0.72  0.66   Globulin, g/L  18.7  19.9  19.9  1.24  0.84   Albumin/globulin ratio  0.63b  0.63b  0.66a  0.01  0.02   Total protein, g/L  30.6  32.5  32.8  1.95  0.99   Urea nitrogen, mmol/L  0.48  0.50  0.44  0.04  0.95   Triglycerides, mmol/L  0.99  0.43  0.76  0.14  0.22   Total cholesterol, mmol/L  4.57a  3.62b  3.42b  0.21  0.05   LDL-C, mmol/L2  0.52a  0.38b  0.43b  0.03  0.05   HDL-C, mmol/L2  1.53  1.62  1.61  0.07  0.56  d 22 to 42   Glucose, mmol/L  14.5b  18.6a  16.9a  0.63  < 0.01   Albumin, g/L  11.6  12.8  12.6  0.39  0.22   Globulin, g/L  16.2  22.0  20.8  1.75  0.14   Albumin/globulin ratio  0.73  0.60  0.61  0.05  0.23   Total protein, g/L  27.8d  34.8c  33.4c  1.95  0.09   Urea nitrogen, mmol/L  0.40a  0.23b  0.34a  0.02  < 0.01   Triglycerides, mmol/L  0.77  0.70  0.50  0.09  0.46   Total cholesterol, mmol/L  3.30c  2.73d  2.70d  0.16  0.08   LDL-C, mmol/L  0.57c  0.34d  0.42c,d  0.05  0.07   HDL-C, mmol/L  1.13d  1.25c,d  1.40c  0.09  0.09  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol. View Large Table 6. Effects of DHA-rich microalgae on serological index of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  d 1 to 21   Glucose, mmol/L  17.6b  18.7a,b  19.6a  0.43  0.03   Albumin, g/L  11.9  12.6  12.9  0.72  0.66   Globulin, g/L  18.7  19.9  19.9  1.24  0.84   Albumin/globulin ratio  0.63b  0.63b  0.66a  0.01  0.02   Total protein, g/L  30.6  32.5  32.8  1.95  0.99   Urea nitrogen, mmol/L  0.48  0.50  0.44  0.04  0.95   Triglycerides, mmol/L  0.99  0.43  0.76  0.14  0.22   Total cholesterol, mmol/L  4.57a  3.62b  3.42b  0.21  0.05   LDL-C, mmol/L2  0.52a  0.38b  0.43b  0.03  0.05   HDL-C, mmol/L2  1.53  1.62  1.61  0.07  0.56  d 22 to 42   Glucose, mmol/L  14.5b  18.6a  16.9a  0.63  < 0.01   Albumin, g/L  11.6  12.8  12.6  0.39  0.22   Globulin, g/L  16.2  22.0  20.8  1.75  0.14   Albumin/globulin ratio  0.73  0.60  0.61  0.05  0.23   Total protein, g/L  27.8d  34.8c  33.4c  1.95  0.09   Urea nitrogen, mmol/L  0.40a  0.23b  0.34a  0.02  < 0.01   Triglycerides, mmol/L  0.77  0.70  0.50  0.09  0.46   Total cholesterol, mmol/L  3.30c  2.73d  2.70d  0.16  0.08   LDL-C, mmol/L  0.57c  0.34d  0.42c,d  0.05  0.07   HDL-C, mmol/L  1.13d  1.25c,d  1.40c  0.09  0.09  Item  CON1  1MA1  2MA1  SEM  P-value  d 1 to 21   Glucose, mmol/L  17.6b  18.7a,b  19.6a  0.43  0.03   Albumin, g/L  11.9  12.6  12.9  0.72  0.66   Globulin, g/L  18.7  19.9  19.9  1.24  0.84   Albumin/globulin ratio  0.63b  0.63b  0.66a  0.01  0.02   Total protein, g/L  30.6  32.5  32.8  1.95  0.99   Urea nitrogen, mmol/L  0.48  0.50  0.44  0.04  0.95   Triglycerides, mmol/L  0.99  0.43  0.76  0.14  0.22   Total cholesterol, mmol/L  4.57a  3.62b  3.42b  0.21  0.05   LDL-C, mmol/L2  0.52a  0.38b  0.43b  0.03  0.05   HDL-C, mmol/L2  1.53  1.62  1.61  0.07  0.56  d 22 to 42   Glucose, mmol/L  14.5b  18.6a  16.9a  0.63  < 0.01   Albumin, g/L  11.6  12.8  12.6  0.39  0.22   Globulin, g/L  16.2  22.0  20.8  1.75  0.14   Albumin/globulin ratio  0.73  0.60  0.61  0.05  0.23   Total protein, g/L  27.8d  34.8c  33.4c  1.95  0.09   Urea nitrogen, mmol/L  0.40a  0.23b  0.34a  0.02  < 0.01   Triglycerides, mmol/L  0.77  0.70  0.50  0.09  0.46   Total cholesterol, mmol/L  3.30c  2.73d  2.70d  0.16  0.08   LDL-C, mmol/L  0.57c  0.34d  0.42c,d  0.05  0.07   HDL-C, mmol/L  1.13d  1.25c,d  1.40c  0.09  0.09  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol. View Large Fatty Acid in Meat Effects of MA on fatty acid content in breast and thigh muscle for birds are shown in Tables 7 and 8. In breast muscle (Table 7), birds fed with MA had increased (P < 0.05) concentration of C14:0, C17:0, C20:5n-3, C22:6n-3, SFA, and n-3 PUFA, and decreased (P < 0.01) content of C18:2n-6, n-6 PUFA, n-6/n-3 PUFA ratio, and P/S compared with CON. Meanwhile, birds fed with 2MA showed higher (P < 0.05) concentration of C16:0, whereas lower (P < 0.05) content of C18:3n-3 and a trend for decreased (P = 0.06) concentration of C18:3n-6 in breast muscle compared with CON. In thigh muscle (Table 8), birds fed with MA had increased (P < 0.05) concentration of C14:0, C20:5n-3, C22:6n-3, and n-3 PUFA, and decreased (P < 0.01) content of C18:2n-6, C18:3n-6, C18:3n-3, n-6/n-3 PUFA ratio, and P/S compared with CON. Meanwhile, birds fed with 2MA showed lower (P < 0.05) content of n-6 PUFA, while birds supplemented with 1MA had decreased (P < 0.05) concentration of C20:0 in thigh muscle compared with CON. Table 7. Effects of DHA-rich microalgae on fatty acid of breast muscle for broilers (g/100 g fresh muscle). Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.47b  0.63a  0.74a  0.04  0.01  C16:0 Palmitic acid  21.8b  23.7a,b  24.9a  0.59  0.03  C16:1n-7 Palmitoleic acid  4.18  4.13  4.67  0.26  0.12  C17:0 Heptadecanoic acid  0.12b  0.15a  0.16a  0.01  0.02  C18:0 Stearic acid  8.40  8.29  8.70  0.56  0.99  C18:1n-9 Oleic acid  31.9  30.5  29.1  1.13  0.34  C18:2n-6 Linoleic acid  25.6a  22.5b  18.6c  0.51  < 0.01  C18:3n-6 Methyl linolenate  0.28d  0.22d,e  0.18e  0.02  0.06  C18:3n-3 α-Linolenic acid  2.02a  1.72a  1.19b  0.12  0.01  C20:0 Arachidic acid  0.12  0.10  0.12  0.01  0.33  C20:1n-9 Twitocene-enoic acid  0.25  0.28  0.27  0.02  0.70  C20:4n-6 Arachidonic acid  2.46  2.44  2.50  0.56  1.00  C20:5n-3 Eicosapentaenoic acid  0.12c  0.34b  0.61a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.30c  2.75b  5.71a  0.62  < 0.01  Saturated fatty acids  30.9c  32.8b  34.6a  0.44  < 0.01  Polyunsaturated fatty acids  30.8  29.9  28.8  0.79  0.17  n-6 Polyunsaturated fatty acids  28.4a  25.1b  21.3c  0.62  < 0.01  n-3 Polyunsaturated fatty acids  2.44c  4.81b  7.52a  0.53  < 0.01  n-6/n-32  11.7a  5.22b  2.94c  0.29  < 0.01  P/S2  1.00a  0.91b  0.83c  0.02  < 0.01  Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.47b  0.63a  0.74a  0.04  0.01  C16:0 Palmitic acid  21.8b  23.7a,b  24.9a  0.59  0.03  C16:1n-7 Palmitoleic acid  4.18  4.13  4.67  0.26  0.12  C17:0 Heptadecanoic acid  0.12b  0.15a  0.16a  0.01  0.02  C18:0 Stearic acid  8.40  8.29  8.70  0.56  0.99  C18:1n-9 Oleic acid  31.9  30.5  29.1  1.13  0.34  C18:2n-6 Linoleic acid  25.6a  22.5b  18.6c  0.51  < 0.01  C18:3n-6 Methyl linolenate  0.28d  0.22d,e  0.18e  0.02  0.06  C18:3n-3 α-Linolenic acid  2.02a  1.72a  1.19b  0.12  0.01  C20:0 Arachidic acid  0.12  0.10  0.12  0.01  0.33  C20:1n-9 Twitocene-enoic acid  0.25  0.28  0.27  0.02  0.70  C20:4n-6 Arachidonic acid  2.46  2.44  2.50  0.56  1.00  C20:5n-3 Eicosapentaenoic acid  0.12c  0.34b  0.61a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.30c  2.75b  5.71a  0.62  < 0.01  Saturated fatty acids  30.9c  32.8b  34.6a  0.44  < 0.01  Polyunsaturated fatty acids  30.8  29.9  28.8  0.79  0.17  n-6 Polyunsaturated fatty acids  28.4a  25.1b  21.3c  0.62  < 0.01  n-3 Polyunsaturated fatty acids  2.44c  4.81b  7.52a  0.53  < 0.01  n-6/n-32  11.7a  5.22b  2.94c  0.29  < 0.01  P/S2  1.00a  0.91b  0.83c  0.02  < 0.01  SEM, standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). d-eDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Table 7. Effects of DHA-rich microalgae on fatty acid of breast muscle for broilers (g/100 g fresh muscle). Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.47b  0.63a  0.74a  0.04  0.01  C16:0 Palmitic acid  21.8b  23.7a,b  24.9a  0.59  0.03  C16:1n-7 Palmitoleic acid  4.18  4.13  4.67  0.26  0.12  C17:0 Heptadecanoic acid  0.12b  0.15a  0.16a  0.01  0.02  C18:0 Stearic acid  8.40  8.29  8.70  0.56  0.99  C18:1n-9 Oleic acid  31.9  30.5  29.1  1.13  0.34  C18:2n-6 Linoleic acid  25.6a  22.5b  18.6c  0.51  < 0.01  C18:3n-6 Methyl linolenate  0.28d  0.22d,e  0.18e  0.02  0.06  C18:3n-3 α-Linolenic acid  2.02a  1.72a  1.19b  0.12  0.01  C20:0 Arachidic acid  0.12  0.10  0.12  0.01  0.33  C20:1n-9 Twitocene-enoic acid  0.25  0.28  0.27  0.02  0.70  C20:4n-6 Arachidonic acid  2.46  2.44  2.50  0.56  1.00  C20:5n-3 Eicosapentaenoic acid  0.12c  0.34b  0.61a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.30c  2.75b  5.71a  0.62  < 0.01  Saturated fatty acids  30.9c  32.8b  34.6a  0.44  < 0.01  Polyunsaturated fatty acids  30.8  29.9  28.8  0.79  0.17  n-6 Polyunsaturated fatty acids  28.4a  25.1b  21.3c  0.62  < 0.01  n-3 Polyunsaturated fatty acids  2.44c  4.81b  7.52a  0.53  < 0.01  n-6/n-32  11.7a  5.22b  2.94c  0.29  < 0.01  P/S2  1.00a  0.91b  0.83c  0.02  < 0.01  Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.47b  0.63a  0.74a  0.04  0.01  C16:0 Palmitic acid  21.8b  23.7a,b  24.9a  0.59  0.03  C16:1n-7 Palmitoleic acid  4.18  4.13  4.67  0.26  0.12  C17:0 Heptadecanoic acid  0.12b  0.15a  0.16a  0.01  0.02  C18:0 Stearic acid  8.40  8.29  8.70  0.56  0.99  C18:1n-9 Oleic acid  31.9  30.5  29.1  1.13  0.34  C18:2n-6 Linoleic acid  25.6a  22.5b  18.6c  0.51  < 0.01  C18:3n-6 Methyl linolenate  0.28d  0.22d,e  0.18e  0.02  0.06  C18:3n-3 α-Linolenic acid  2.02a  1.72a  1.19b  0.12  0.01  C20:0 Arachidic acid  0.12  0.10  0.12  0.01  0.33  C20:1n-9 Twitocene-enoic acid  0.25  0.28  0.27  0.02  0.70  C20:4n-6 Arachidonic acid  2.46  2.44  2.50  0.56  1.00  C20:5n-3 Eicosapentaenoic acid  0.12c  0.34b  0.61a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.30c  2.75b  5.71a  0.62  < 0.01  Saturated fatty acids  30.9c  32.8b  34.6a  0.44  < 0.01  Polyunsaturated fatty acids  30.8  29.9  28.8  0.79  0.17  n-6 Polyunsaturated fatty acids  28.4a  25.1b  21.3c  0.62  < 0.01  n-3 Polyunsaturated fatty acids  2.44c  4.81b  7.52a  0.53  < 0.01  n-6/n-32  11.7a  5.22b  2.94c  0.29  < 0.01  P/S2  1.00a  0.91b  0.83c  0.02  < 0.01  SEM, standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). d-eDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Table 8. Effects of DHA-rich microalgae on fatty acid of thigh muscle for broilers (g/100 g fresh muscle). Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.49b  0.63a  0.64a  0.03  0.02  C16:0 Palmitic acid  24.4  23.4  23.3  1.70  0.86  C16:1n-7 Palmitoleic acid  4.55  3.80  3.54  0.45  0.31  C17:0 Heptadecanoic acid  0.13  0.16  0.16  0.01  0.36  C18:0 Stearic acid  9.46  8.84  9.77  0.93  0.50  C18:1n-9 Oleic acid  22.7  30.0  28.9  6.36  0.62  C18:2n-6 Linoleic acid  28.0a  21.7b  16.1b  1.74  0.01  C18:3n-6 Methyl linolenate  0.33a  0.23b  0.21b  0.02  0.04  C18:3n-3 α-Linolenic acid  2.25a  1.62b  0.93c  0.11  < 0.01  C20:0 Arachidic acid  0.18a  0.13b  0.17a  0.01  0.04  C20:1n-9 Twitocene-enoic acid  0.30  0.25  0.29  0.06  0.60  C20:4n-6 Arachidonic acid  3.51  2.93  3.09  0.78  0.79  C20:5n-3 Eicosapentaenoic acid  0.24c  0.35b  0.54a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.43c  3.44b  8.64a  0.21  < 0.01  Saturated fatty acids  34.7  33.2  34.1  2.59  0.82  Polyunsaturated fatty acids  34.8  30.2  29.5  2.69  0.35  n-6 Polyunsaturated fatty acids  31.8a  24.8a,b  19.4b  2.52  0.04  n-3 Polyunsaturated fatty acids  2.92c  5.41b  10.1a  0.27  < 0.01  n-6/n-32  10.9a  4.62b  1.92c  0.38  < 0.01  P/S2  1.00a  0.91b  0.87b  0.02  0.01  Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.49b  0.63a  0.64a  0.03  0.02  C16:0 Palmitic acid  24.4  23.4  23.3  1.70  0.86  C16:1n-7 Palmitoleic acid  4.55  3.80  3.54  0.45  0.31  C17:0 Heptadecanoic acid  0.13  0.16  0.16  0.01  0.36  C18:0 Stearic acid  9.46  8.84  9.77  0.93  0.50  C18:1n-9 Oleic acid  22.7  30.0  28.9  6.36  0.62  C18:2n-6 Linoleic acid  28.0a  21.7b  16.1b  1.74  0.01  C18:3n-6 Methyl linolenate  0.33a  0.23b  0.21b  0.02  0.04  C18:3n-3 α-Linolenic acid  2.25a  1.62b  0.93c  0.11  < 0.01  C20:0 Arachidic acid  0.18a  0.13b  0.17a  0.01  0.04  C20:1n-9 Twitocene-enoic acid  0.30  0.25  0.29  0.06  0.60  C20:4n-6 Arachidonic acid  3.51  2.93  3.09  0.78  0.79  C20:5n-3 Eicosapentaenoic acid  0.24c  0.35b  0.54a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.43c  3.44b  8.64a  0.21  < 0.01  Saturated fatty acids  34.7  33.2  34.1  2.59  0.82  Polyunsaturated fatty acids  34.8  30.2  29.5  2.69  0.35  n-6 Polyunsaturated fatty acids  31.8a  24.8a,b  19.4b  2.52  0.04  n-3 Polyunsaturated fatty acids  2.92c  5.41b  10.1a  0.27  < 0.01  n-6/n-32  10.9a  4.62b  1.92c  0.38  < 0.01  P/S2  1.00a  0.91b  0.87b  0.02  0.01  SEM, standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Table 8. Effects of DHA-rich microalgae on fatty acid of thigh muscle for broilers (g/100 g fresh muscle). Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.49b  0.63a  0.64a  0.03  0.02  C16:0 Palmitic acid  24.4  23.4  23.3  1.70  0.86  C16:1n-7 Palmitoleic acid  4.55  3.80  3.54  0.45  0.31  C17:0 Heptadecanoic acid  0.13  0.16  0.16  0.01  0.36  C18:0 Stearic acid  9.46  8.84  9.77  0.93  0.50  C18:1n-9 Oleic acid  22.7  30.0  28.9  6.36  0.62  C18:2n-6 Linoleic acid  28.0a  21.7b  16.1b  1.74  0.01  C18:3n-6 Methyl linolenate  0.33a  0.23b  0.21b  0.02  0.04  C18:3n-3 α-Linolenic acid  2.25a  1.62b  0.93c  0.11  < 0.01  C20:0 Arachidic acid  0.18a  0.13b  0.17a  0.01  0.04  C20:1n-9 Twitocene-enoic acid  0.30  0.25  0.29  0.06  0.60  C20:4n-6 Arachidonic acid  3.51  2.93  3.09  0.78  0.79  C20:5n-3 Eicosapentaenoic acid  0.24c  0.35b  0.54a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.43c  3.44b  8.64a  0.21  < 0.01  Saturated fatty acids  34.7  33.2  34.1  2.59  0.82  Polyunsaturated fatty acids  34.8  30.2  29.5  2.69  0.35  n-6 Polyunsaturated fatty acids  31.8a  24.8a,b  19.4b  2.52  0.04  n-3 Polyunsaturated fatty acids  2.92c  5.41b  10.1a  0.27  < 0.01  n-6/n-32  10.9a  4.62b  1.92c  0.38  < 0.01  P/S2  1.00a  0.91b  0.87b  0.02  0.01  Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.49b  0.63a  0.64a  0.03  0.02  C16:0 Palmitic acid  24.4  23.4  23.3  1.70  0.86  C16:1n-7 Palmitoleic acid  4.55  3.80  3.54  0.45  0.31  C17:0 Heptadecanoic acid  0.13  0.16  0.16  0.01  0.36  C18:0 Stearic acid  9.46  8.84  9.77  0.93  0.50  C18:1n-9 Oleic acid  22.7  30.0  28.9  6.36  0.62  C18:2n-6 Linoleic acid  28.0a  21.7b  16.1b  1.74  0.01  C18:3n-6 Methyl linolenate  0.33a  0.23b  0.21b  0.02  0.04  C18:3n-3 α-Linolenic acid  2.25a  1.62b  0.93c  0.11  < 0.01  C20:0 Arachidic acid  0.18a  0.13b  0.17a  0.01  0.04  C20:1n-9 Twitocene-enoic acid  0.30  0.25  0.29  0.06  0.60  C20:4n-6 Arachidonic acid  3.51  2.93  3.09  0.78  0.79  C20:5n-3 Eicosapentaenoic acid  0.24c  0.35b  0.54a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.43c  3.44b  8.64a  0.21  < 0.01  Saturated fatty acids  34.7  33.2  34.1  2.59  0.82  Polyunsaturated fatty acids  34.8  30.2  29.5  2.69  0.35  n-6 Polyunsaturated fatty acids  31.8a  24.8a,b  19.4b  2.52  0.04  n-3 Polyunsaturated fatty acids  2.92c  5.41b  10.1a  0.27  < 0.01  n-6/n-32  10.9a  4.62b  1.92c  0.38  < 0.01  P/S2  1.00a  0.91b  0.87b  0.02  0.01  SEM, standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Antioxidant Capacity Effects of MA on antioxidant capacity of broilers are shown in Table 9. In breast muscle, birds fed with 1MA showed greater (P < 0.05) concentration of SOD, CAT, and a trend of improved content of T-AOC (P = 0.07), while they also had a lower (P < 0.05) level of MDA compared with CON. Birds supplemented with 2MA had improved (P < 0.05) content of SOD compared with CON. In thigh muscle, the content of T-AOC and SOD was higher (P < 0.05) in birds fed with 1MA, while the concentration of GSH-Px was greater (P < 0.05) in birds fed with 2MA, and the content of MDA tended to be lower (P = 0.09) in birds supplemented with 1MA compared with CON. Table 9. Effects of DHA-rich microalgae on antioxidant capacity in breast and thigh muscle of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle:   Superoxide dismutase, U/mg  114b  162a  158a  10.9  0.05   Total antioxidant capacity, U/mg  5.63d  9.90c  9.01c,d  0.97  0.07   Glutathione peroxidase, U/mg  699  925  855  63.3  0.14   Catalase, U/mg  5.26b  9.78a  5.82b  0.80  0.03   Malondialdehyde, nmol/mg  5.64a  3.67b  4.35a,b  0.41  0.05  Thigh muscle:   Superoxide dismutase, U/mg  118b  185a  163a,b  16.1  0.05   Total antioxidant capacity, U/mg  5.71b  10.36a  7.61b  0.53  < 0.01   Glutathione peroxidase, U/mg  661b  853a,b  964a  55.2  0.04   Catalase, U/mg  6.36  9.25  6.15  0.91  0.13   Malondialdehyde, nmol/mg  4.67c  3.56d  4.16c,d  0.26  0.09  Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle:   Superoxide dismutase, U/mg  114b  162a  158a  10.9  0.05   Total antioxidant capacity, U/mg  5.63d  9.90c  9.01c,d  0.97  0.07   Glutathione peroxidase, U/mg  699  925  855  63.3  0.14   Catalase, U/mg  5.26b  9.78a  5.82b  0.80  0.03   Malondialdehyde, nmol/mg  5.64a  3.67b  4.35a,b  0.41  0.05  Thigh muscle:   Superoxide dismutase, U/mg  118b  185a  163a,b  16.1  0.05   Total antioxidant capacity, U/mg  5.71b  10.36a  7.61b  0.53  < 0.01   Glutathione peroxidase, U/mg  661b  853a,b  964a  55.2  0.04   Catalase, U/mg  6.36  9.25  6.15  0.91  0.13   Malondialdehyde, nmol/mg  4.67c  3.56d  4.16c,d  0.26  0.09  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. View Large Table 9. Effects of DHA-rich microalgae on antioxidant capacity in breast and thigh muscle of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle:   Superoxide dismutase, U/mg  114b  162a  158a  10.9  0.05   Total antioxidant capacity, U/mg  5.63d  9.90c  9.01c,d  0.97  0.07   Glutathione peroxidase, U/mg  699  925  855  63.3  0.14   Catalase, U/mg  5.26b  9.78a  5.82b  0.80  0.03   Malondialdehyde, nmol/mg  5.64a  3.67b  4.35a,b  0.41  0.05  Thigh muscle:   Superoxide dismutase, U/mg  118b  185a  163a,b  16.1  0.05   Total antioxidant capacity, U/mg  5.71b  10.36a  7.61b  0.53  < 0.01   Glutathione peroxidase, U/mg  661b  853a,b  964a  55.2  0.04   Catalase, U/mg  6.36  9.25  6.15  0.91  0.13   Malondialdehyde, nmol/mg  4.67c  3.56d  4.16c,d  0.26  0.09  Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle:   Superoxide dismutase, U/mg  114b  162a  158a  10.9  0.05   Total antioxidant capacity, U/mg  5.63d  9.90c  9.01c,d  0.97  0.07   Glutathione peroxidase, U/mg  699  925  855  63.3  0.14   Catalase, U/mg  5.26b  9.78a  5.82b  0.80  0.03   Malondialdehyde, nmol/mg  5.64a  3.67b  4.35a,b  0.41  0.05  Thigh muscle:   Superoxide dismutase, U/mg  118b  185a  163a,b  16.1  0.05   Total antioxidant capacity, U/mg  5.71b  10.36a  7.61b  0.53  < 0.01   Glutathione peroxidase, U/mg  661b  853a,b  964a  55.2  0.04   Catalase, U/mg  6.36  9.25  6.15  0.91  0.13   Malondialdehyde, nmol/mg  4.67c  3.56d  4.16c,d  0.26  0.09  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. View Large DISCUSSION In the present research, we demonstrated that birds fed diets supplemented with 1% and 2% MA showed improved BW, ADG, and lower FCR, which is in agreement with Kessi (2016). This result may be due to the positive effects of MA on improving serum composition and immune status (Abudabos et al., 2013), and decreasing microbial load in digestive tract (Zahid et al., 2001; Ali and Memon, 2008) of birds. However, birds fed with 2MA showed decreased ADFI even though ADG was increased, which might be related to the high content of fiber (polysaccharide) in the algal biomass (Armin et al., 2015), the high concentrations of phenolic compounds (De Lange, 2000), or the high levels of heavy metals, toxins, and arsenic in MA (Harinder et al., 2016). The improved thigh muscle weight and yield in our study is associated with the higher final BW of birds by MA (Abudabos et al., 2013; Ribeiro et al., 2013). However, unlike our study, Bharath et al. (2017) reported carcass traits like dressing yield, breast yield and liver percentage were not influenced by n-3 PUFA, which might be due to the different type and amount of MA used (Harinder et al., 2016). The decreased abdominal fat percentage by MA in our study is due to the positive effects of DHA in MA on utilization of lipid in serum (Gatrell et al., 2017), which is usually revealed by the decreased serum cholesterol and triglyceride levels (Armin et al., 2015), while our study also found the concentration of LDL-C in serum was reduced. The improved liver percentage and spleen percentage in our study were beneficial for the immunity in birds and n-3 PUFA deposition in chicken meat (Mariey et al., 2014), which might result in better growth performance. Moreover, these improved liver percentage and spleen percentage might be the reason for the increased serological index and higher content of n-3 PUFA in diets and muscle (Schreiner et al., 2005). Together with the improved carcass trait, birds also showed better serological index. Serum glucose is an important source of energy for animals and can be conducive to growth of body tissues, while serum concentrations of albumin and total protein reflect the functions of synthesis of proteins in liver of broilers, which may associate with animal growth and physiological status (Limdi and Hyde, 2003). In the present study, birds fed MA showed increased levels of serum glucose, total protein, albumin, and globulin, which was also revealed by Mariey et al. (2014). Furthermore, the MA-induced reduction in serum UN observed in our study suggests that MA helped birds achieve more efficient nitrogen utilization (Brown and Cline, 1974), while this nitrogen retention or nitrogen balance helps to the balance between body protein synthesis and body protein degradation in birds (Metayer et al., 2008). Except for the glucose and protein metabolism, the lipid utilization was also improved in our study, which were also revealed in the studies of Abudabos et al. (2013) and Mariey et al. (2014). In our study, birds fed with MA showed the lower fat content in serum of birds, especially TC, TG, and LDL-C (Wahbeh, 1997; Abudabos et al., 2013), which was associated with higher concentration of n-3 PUFA in diets and muscle, particularly EPA and DHA. Actually, DHA supplementation from MA reduced serum TG and increase HDL-C (Bernstein et al., 2011) because DHA can decrease TG levels by reducing hepatic very low-density lipoprotein (VLDL) synthesis (Roche and Gibney, 2000). Reduced synthesis in turn lead to reduced secretion and smaller VLDL particles, which were more readily converted to LDL and HDL (Mori et al., 2000). Cholesterol issues were usually addressed by lowering LDL-C, while increasing concentration of serum HDL-C was considered as an important therapeutic target for improvement of the lipid profile and some cardiovascular disease (Colla et al., 2008). Our results showed MA regulated blood lipid balance might be through promoting HDL-C synthesis and accelerating TC, TG, LDL-C metabolism. The improved serum composition in birds might help reduce the risk for cardiovascular disease in persons who consume the chicken meats from broilers fed diets containing MA. Along with the altered serological index, we found the antioxidant capacity and fatty acid concentration in breast and thigh meat were also increased, which is in line with Armin et al. (2015), who found that 1% MA elicited an enhancement in n-3 PUFA accumulation on thigh and breast meat of birds along with improved antioxidant status due to the positive effect of high level of DHA in MA. Our results showed MA increased the concentrations of C14:0, C16:0, and SFA in meat, which is possibly because MA contain higher concentrations of C14:0 (5.15%), C16:0 (60.1%), and SFA (67.9%) than SO, while C14:0, C16:0, and SFA are hard to be oxidant and easy to be deposited. The PUFA in MA is 29%, lower than SO (62%), but the n-3 PUFA in MA is 29%, higher than SO (8%), which indicates MA mainly contains n-3 PUFA, while SO mainly contains n-6 PUFA. In our study, we also found MA could lower the concentration of n-6 PUFA in diets, breast and thigh muscle of birds because MA contain nearly no n-6 PUFA. This chicken meat might be beneficial for humans who consume it because a higher intake of n-6 PUFA in meat might increase the risk of human gallstones (Sturdevant et al., 1973), reduce high-density lipoprotein concentrations (Mattson and Grundy, 1985), and inhibit immune system function (Rasmussen et al., 1994). The most important n-3 PUFA in human nutrition are α-Linolenic acid (C18:3n-3), EPA, and DHA (Burdge, 2004). Our study found that α-Linolenic acid were reduced along with the increased level of EPA and DHA, which is because α-Linolenic acid serves as a precursor for the synthesis of EPA and DHA in thigh and breast muscle of broilers. Considering this conversion is limited in humans (Garg et al., 2006), our study uses MA (containing 28.7% DHA) as a primary source of DHA to improve the concentration of DHA in chicken meat (Lopez-Ferrer et al., 2001a; Zelenka et al., 2008). Our study demonstrates that the fatty acid profiles in breast or thigh muscle are in line with these in diets, which is because broilers are normally in a positive energy balance and the PUFAs supplied through diets are deposited in tissues mostly unaltered, so the composition of carcass fat tends to reflect that of dietary fat (Lopez-Ferrer et al., 2001a; Betti et al., 2009; Ribeiro et al., 2013). The key to positive health effects of n-3 PUFA was a balanced ratio of n-6 to n-3 PUFA (Schreiner et al., 2005), which might reduce the risk of lifestyle diseases such as coronary artery disease. Burghardt et al. (2010) pointed out nutritional recommendations in human diets are that the P/S ratio should be above 0.45 and the n-6/n-3 PUFA ratio should not exceed 4, while Sugano (1996) found the recommended ratio of n-6/n-3 PUFA ratio in the human diets ranges between 3:1 and 6:1. Interestingly, these requirements are comparable to the ratios obtained in the breast and thigh meat of chickens fed MA in our study, so consumption of this chicken meat by humans might help reduce the risk of coronary artery atherosclerotic disease and some common adult cancers (Renehan et al., 2008; Jiang et al., 2013). Due to the nutritional and physiological characteristics, broilers are prone to lipid peroxidation, resulting in more peroxidative metabolites. Feeding birds with lipid-rich diet may cause oxidative stress, which may damage DNA, bio-membrane lipids, and proteins, as well as a variety of impairments to tissue of birds (Zhao and Shen, 2005; Zhang et al., 2011; Delles et al., 2014). The MDA is a major product of lipid peroxidation, and is an effective marker of oxidative stress (Lu et al., 2010). In our research, we found 1% or 2% MA was effective in decreasing level of MDA in meat mainly because of the DHA and antioxidants in MA. For one thing, the high concentration of DHA scavenges free radical and regulate the level of reactive oxygen species in vivo through the action of NAD (P) H oxidase or through its own peroxidation reaction and free radical reaction (Richard et al., 2008). The present study demonstrates DHA in MA reduce MDA via activing the enzyme and the non-enzyme antioxidant system. Enzymatic inactivation of reactive oxygen species in muscle tissue is mainly achieved by the higher concentration of SOD, CAT, and GSH-Px because SOD and CAT are antioxidant enzymes that directly react with radical species, whereas GSH-Px regenerates oxidized antioxidants (Delles et al., 2014). The non-enzymatic antioxidant defense system is reflected by the increased concentration of T-AOC (Wang et al., 2011). For another, MA had some antioxidants, including beta-carotene and vitamin E, which may play an important role in protecting endogenous lipids from peroxidation and oxidation. Our findings suggest that 1% or 2% MA (containing 2.04% vitamin A and 0.07% vitamin E) can increase antioxidant capacity of chicken meat. The vitamin A in our study may reflect the function of beta-carotene on producing high-oxidative stability chicken meat (Barclay et al., 1994) because beta-carotene is one of the provitamin A carotenoids (Olson, 1989). Furthermore, the MA in our study may have the same effect as vitamin E in lowering concentration of MDA and improving oxidative stability of broiler meat enriched with n-3 PUFA (Rymer et al., 2010; Armin et al., 2015). CONCLUSION These results demonstrate that supplementing diets with 1% or 2% microalgae (Schizochytrium limacinum CCAP 4087/2) replacing soybean oil effectively improved performance, carcass traits, serum composition, antioxidant capacity, and n-3 PUFA (EPA and DHA) deposition in breast and thigh muscle of broiler chickens. ACKNOWLEDGMENTS This research is supported by the National Natural Science Foundation of China (31772612) and CARS-35. We also acknowledge Alltech Inc. (Nicholasville, KY) for providing the novel microalgae products which enriches DHA. REFERENCES Abudabos A. M., Okab A. B., Aljumaah R. S., Samara E. M.. 2013. Nutritional value of green seaweed (ulva lactuca) for broiler chickens. Ital. J. Anim. Sci.  12: 612– 620. Adkins Y., Kelley D. S.. 2010. 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Dietary supplementation with DHA-rich microalgae improves performance, serum composition, carcass trait, antioxidant status, and fatty acid profile of broilers

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© 2018 Poultry Science Association Inc.
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ABSTRACT This experiment was conducted with 126 as-hatched male Arbor Acres chicks (1-d-old, weighing 45.3 ± 0.72 g) to determine the effects of microalgae [MA, containing 29% docosahexaenoic acid (DHA)] on performance, serum composition, carcass trait, antioxidant status, and fatty acid deposition of birds. The birds were allocated randomly to 1 of 3 treatments with 7 replicate pens per treatment (6 birds per pen). The dietary treatments included a control diet [corn-soybean basal diet supplemented with 3% soybean oil (SO), CON], 1% MA diet (basal diet supplemented with 1% MA and 2% SO, 1MA), and 2% MA diet (basal diet supplemented with 2% MA and 1% SO, 2MA). All birds were raised in wire-floored cages. The trial consists of a starter phase from d 1 to 21 and a grower phase from d 22 to 42. Compared with CON, birds supplemented with MA (1MA or 2MA) had greater (P < 0.05) average daily gain, liver percentage (liver weight/body weight), and serum glucose, as well as lower (P < 0.05) feed conversation ratio, abdominal fat percentage (abdominal fat weight/body weight), and total serum cholesterol. Moreover, due to the high concentration of DHA in MA, birds fed MA showed increased (P < 0.05) concentration of eicosapentaenoic acid, DHA, superoxide dismutase, and total antioxidant capacity, as well as decreased (P < 0.05) n-6/n-3 polyunsaturated fatty acid ratio, polyunsaturated fatty acid/saturated fatty acid ratio, and malondialdehyde in the breast and thigh muscle compared with CON. In conclusion, dietary supplementation with 1% or 2% DHA-rich microalgae had positive effects on performance, serum composition, carcass trait, antioxidant status, and fatty acid deposition in birds. INTRODUCTION Dietary intakes of high level long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), particularly eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), are suboptimal in humans’ diets (Givens and Gibbs, 2008; Adkins and Kelley, 2010). However, LC n-3 PUFA and DHA are crucial for decreasing risk of cancer, atherosclerosis, cardiovascular disease, coronary heart disease, and other related diseases in humans (Singh et al., 1997; Marckmann and Gronbæk, 1999; Zhang et al., 2010) because they can help to reduce concentrations of serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (Bang and Dyerberg, 1972; Abudabos et al., 2013). Researchers have reported that DHA is rich in marine fish and some marine plants, especially some strains of microalgae (MA) et al., 2016). One efficient approach to increase intakes of DHA is via the enrichment of basic foods for humans (Alasalvar et al., 2002; Arterburn et al., 2006; Schmitz and Ecker, 2008). Since poultry meat has become one of the most consumed meats with an annual average per capita consumption of 22 kg in Europe (Eurostat, 2008) and 39 kg in the United States (USDA, 2010), scientists have focused on manipulating fatty acid composition of poultry meat through altering fatty acid composition of broiler diets, with the goal of increasing DHA consumption in human diets (Lopez-Ferrer et al., 2001a; Rymer and Givens, 2005; Givens and Gibbs, 2008). Supplementation of animal diets with lipids and oils rich in n-3 PUFA is regarded as an efficient way to improve the concentration of DHA and EPA in animal tissues (Lopez-Ferrer et al., 2001b). Usually, the enrichment of LC n-3 PUFA in poultry meat is realized by adding fish oil to the broiler diet (Rymer and Givens, 2005). However, use of fish oil may reduce oxidative stability of poultry meat (O’Keefe et al., 1995; Bou et al., 2001) because high concentration of PUFA in fish oil is easily to produced off-flavors (Meynier et al., 1999), and off-odors (Kahraman et al., 2004; Wood et al., 2008). An alternative approach is to feed birds with diets containing marine algae to produce high-oxidative stability chicken meat (Mooney et al., 1998; Guschina and Harwood, 2006), because marine algae (the primary producers of DHA) have high content of DHA and some antioxidants, including beta-carotenoids, vitamin A, and vitamin E (Barclay et al., 1994). Low level of Algal biomass (1% to 5%) has been demonstrated to be effective in improving DHA concentration of pig meat (Sardi et al., 2006), eggs (Cheng et al., 2005; Cachaldora et al., 2008), and poultry meat (Mooney et al., 1998; Kralik et al., 2004). Moreover, MA from Schizochytrium sp. are rich in DHA and vitamins (e.g., beta-carotene and vitamin E), and often used as one of the best candidates to produce sustainable and affordable DHA (Hakim, 2013). However, there are few studies focusing on the effects of low level of MA from Schizochytrium sp. on both fatty acid deposition and antioxidant status. So the exact impact of adding low level of MA replacing soybean oil (SO) into broilers’ diets on broiler performance, carcass trait, and lipid profile remains to be established although it is well known that marine algae are excellent sources of DHA. The objective of the experiment reported in this paper was to investigate the effect of adding different levels of a DHA-rich MA strain replacing SO on performance, serum composition, carcass trait, antioxidant status, and deposition of LC n-3 PUFA in broilers. MATERIALS AND MMETHODS All the procedures used in this animal experiment were approved by the Institutional Animal Care and Use Committee of China Agricultural University (Beijing, China). Experimental Product A commercial source of dehydrated, whole-cell MA, All-G-Rich (Schizochytrium limacinum CCAP 4087/2), which contains 64% fat, 29% DHA, 11% crude protein, 2.04% vitamin A, and 0.07% vitamin E, was supplied by Alltech Inc. (Nicholasville, KY). The SO was obtained from Beijing Tongli Xing Department of Agricultural Science and Technology Company Limited (Beijing, China). Fatty acid composition of MA and SO used in the experiment is displayed in Table 1. Table 1. Fatty acid composition of microalgae and soybean oil used in experimental diets (% total fat). Fatty acid  MA1  SO1  C14:0 Myristic acid  5.15  0.08  C16:0 Palmitic acid  60.1  10.9  C16:1n-7 Palmitoleic acid  0.13  0.09  C17:0 Heptadecanoic acid  0.56  0.10  C18:0 Stearic acid  1.79  4.40  C18:1n-9 Oleic acid  0.03  20.1  C18:2n-6 Linoleic acid  0.04  53.5  C18:3n-6 Methyl linolenate  0.08  0.08  C18:3n-3 α-Linolenic acid  0.05  7.86  C20:0 Arachidic acid  0.29  0.00  C20:1n-9 Twitocene-enoic acid  0.12  0.03  C20:4n-6 Arachidonic acid  0.08  0.00  C20:5n-3 Eicosapentaenoic acid  0.30  0.01  C22:6n-3 Docosahexaenoic acid  28.7  0.04  Saturated fatty acids  67.9  15.5  Monounsaturated fatty acids  0.15  20.1  Polyunsaturated fatty acids  29.2  61.5  n-6 Polyunsaturated fatty acids  0.19  53.6  n-3 Polyunsaturated fatty acids  29.1  7.91  n-6/n-32  0.01  6.77  P/S2  0.43  3.97  Fatty acid  MA1  SO1  C14:0 Myristic acid  5.15  0.08  C16:0 Palmitic acid  60.1  10.9  C16:1n-7 Palmitoleic acid  0.13  0.09  C17:0 Heptadecanoic acid  0.56  0.10  C18:0 Stearic acid  1.79  4.40  C18:1n-9 Oleic acid  0.03  20.1  C18:2n-6 Linoleic acid  0.04  53.5  C18:3n-6 Methyl linolenate  0.08  0.08  C18:3n-3 α-Linolenic acid  0.05  7.86  C20:0 Arachidic acid  0.29  0.00  C20:1n-9 Twitocene-enoic acid  0.12  0.03  C20:4n-6 Arachidonic acid  0.08  0.00  C20:5n-3 Eicosapentaenoic acid  0.30  0.01  C22:6n-3 Docosahexaenoic acid  28.7  0.04  Saturated fatty acids  67.9  15.5  Monounsaturated fatty acids  0.15  20.1  Polyunsaturated fatty acids  29.2  61.5  n-6 Polyunsaturated fatty acids  0.19  53.6  n-3 Polyunsaturated fatty acids  29.1  7.91  n-6/n-32  0.01  6.77  P/S2  0.43  3.97  1MA: microalgae; SO: soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Table 1. Fatty acid composition of microalgae and soybean oil used in experimental diets (% total fat). Fatty acid  MA1  SO1  C14:0 Myristic acid  5.15  0.08  C16:0 Palmitic acid  60.1  10.9  C16:1n-7 Palmitoleic acid  0.13  0.09  C17:0 Heptadecanoic acid  0.56  0.10  C18:0 Stearic acid  1.79  4.40  C18:1n-9 Oleic acid  0.03  20.1  C18:2n-6 Linoleic acid  0.04  53.5  C18:3n-6 Methyl linolenate  0.08  0.08  C18:3n-3 α-Linolenic acid  0.05  7.86  C20:0 Arachidic acid  0.29  0.00  C20:1n-9 Twitocene-enoic acid  0.12  0.03  C20:4n-6 Arachidonic acid  0.08  0.00  C20:5n-3 Eicosapentaenoic acid  0.30  0.01  C22:6n-3 Docosahexaenoic acid  28.7  0.04  Saturated fatty acids  67.9  15.5  Monounsaturated fatty acids  0.15  20.1  Polyunsaturated fatty acids  29.2  61.5  n-6 Polyunsaturated fatty acids  0.19  53.6  n-3 Polyunsaturated fatty acids  29.1  7.91  n-6/n-32  0.01  6.77  P/S2  0.43  3.97  Fatty acid  MA1  SO1  C14:0 Myristic acid  5.15  0.08  C16:0 Palmitic acid  60.1  10.9  C16:1n-7 Palmitoleic acid  0.13  0.09  C17:0 Heptadecanoic acid  0.56  0.10  C18:0 Stearic acid  1.79  4.40  C18:1n-9 Oleic acid  0.03  20.1  C18:2n-6 Linoleic acid  0.04  53.5  C18:3n-6 Methyl linolenate  0.08  0.08  C18:3n-3 α-Linolenic acid  0.05  7.86  C20:0 Arachidic acid  0.29  0.00  C20:1n-9 Twitocene-enoic acid  0.12  0.03  C20:4n-6 Arachidonic acid  0.08  0.00  C20:5n-3 Eicosapentaenoic acid  0.30  0.01  C22:6n-3 Docosahexaenoic acid  28.7  0.04  Saturated fatty acids  67.9  15.5  Monounsaturated fatty acids  0.15  20.1  Polyunsaturated fatty acids  29.2  61.5  n-6 Polyunsaturated fatty acids  0.19  53.6  n-3 Polyunsaturated fatty acids  29.1  7.91  n-6/n-32  0.01  6.77  P/S2  0.43  3.97  1MA: microalgae; SO: soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Experimental Animals and Management As-hatched Arbor Acres chicks (n = 126; 1 day of age, weighing 45.3 ± 0.72 g) were purchased from Arbor Acres Poultry Breeding Company (Beijing, China). All birds were raised in wire-floored cages in an environmentally controlled room with continuous light (10 to 20 lux) and were permitted ad libitum access to feed and water. Room temperature was maintained at 33°C for the first 3 d, then the temperature was reduced gradually by 3°C per week until reaching 24°C which was maintained until the end of the 42-d experiment. The lighting regimen and ventilation were monitored continuously from d 1 to 42. All birds were inoculated with Newcastle disease vaccine on d 7 and 28 and with inactivated infectious bursa disease vaccine on d 14 and 21. The trial was conducted in 2 phases consisting of a starter phase from d 1 to 21 (phase 1) and a grower phase from d 22 to 42 (phase 2). Experimental Design and Diets Broilers were allotted randomly to 1 of 3 dietary treatments (Table 2). The dietary treatments included a control diet [corn-soybean basal diet supplemented with 3% soybean oil (SO), CON], 1% MA diet (basal diet supplemented with 1% MA and 2% SO, 1MA), 2% MA diet (basal diet supplemented with 2% MA and 1% SO, 2MA). There were 7 replicate pens per treatment with 6 birds per pen. All essential nutrients contained in the basal diet in phase 1 and phase 2 met the nutrients requirements suggested by the NRC (1994). All diets were fed in mash form. Table 2. Composition and nutrient levels of experimental diets (%, as-fed basis). Item  Phase 1 (d 1 to 21)  Phase 2 (d 22 to 42)    CON1  1MA1  2MA1  CON  1MA  2MA  Corn  58.70  58.70  58.70  65.40  65.40  65.40  Soybean meal  30.11  30.11  30.11  22.69  22.69  22.69  Corn gluten meal  4.00  4.00  4.00  5.10  5.10  5.10  Soybean oil  3.00  2.00  1.00  3.00  2.00  1.00  Microalgae  -  1.00  2.00  -  1.00  2.00  Dicalcium phosphate  1.70  1.70  1.70  1.25  1.25  1.25  Limestone  1.39  1.39  1.39  1.44  1.44  1.44  Salt  0.30  0.30  0.30  0.30  0.30  0.30  L-lysine HCl, 78%  0.12  0.12  0.12  0.21  0.21  0.21  DL-Methionine, 98%  0.15  0.15  0.15  0.05  0.05  0.05  L-Threonine, 98%  0.03  0.03  0.03  0.06  0.06  0.06  Vitamin-mineral Premix2  0.50  0.50  0.50  0.50  0.50  0.50  Nutrient composition              Metabolic Energy, MJ/kg3  12.76  12.69  12.61  13.18  13.12  13.04  Crude Protein4  20.70  20.93  20.46  20.10  19.88  19.86  Calcium3  1.00  1.00  1.00  0.90  0.90  0.90  Available Phosphorus3  0.45  0.45  0.45  0.35  0.35  0.35  SID lysine3  1.10  1.10  1.10  1.00  1.00  1.00  SID methionine3  0.50  0.50  0.50  0.38  0.38  0.38  SID threonine3  0.80  0.80  0.80  0.74  0.74  0.74  SID tryptophan3  0.27  0.27  0.27  0.23  0.23  0.23  Item  Phase 1 (d 1 to 21)  Phase 2 (d 22 to 42)    CON1  1MA1  2MA1  CON  1MA  2MA  Corn  58.70  58.70  58.70  65.40  65.40  65.40  Soybean meal  30.11  30.11  30.11  22.69  22.69  22.69  Corn gluten meal  4.00  4.00  4.00  5.10  5.10  5.10  Soybean oil  3.00  2.00  1.00  3.00  2.00  1.00  Microalgae  -  1.00  2.00  -  1.00  2.00  Dicalcium phosphate  1.70  1.70  1.70  1.25  1.25  1.25  Limestone  1.39  1.39  1.39  1.44  1.44  1.44  Salt  0.30  0.30  0.30  0.30  0.30  0.30  L-lysine HCl, 78%  0.12  0.12  0.12  0.21  0.21  0.21  DL-Methionine, 98%  0.15  0.15  0.15  0.05  0.05  0.05  L-Threonine, 98%  0.03  0.03  0.03  0.06  0.06  0.06  Vitamin-mineral Premix2  0.50  0.50  0.50  0.50  0.50  0.50  Nutrient composition              Metabolic Energy, MJ/kg3  12.76  12.69  12.61  13.18  13.12  13.04  Crude Protein4  20.70  20.93  20.46  20.10  19.88  19.86  Calcium3  1.00  1.00  1.00  0.90  0.90  0.90  Available Phosphorus3  0.45  0.45  0.45  0.35  0.35  0.35  SID lysine3  1.10  1.10  1.10  1.00  1.00  1.00  SID methionine3  0.50  0.50  0.50  0.38  0.38  0.38  SID threonine3  0.80  0.80  0.80  0.74  0.74  0.74  SID tryptophan3  0.27  0.27  0.27  0.23  0.23  0.23  1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). 2Premix supplied per kg diet: vitamin A, 11,000 IU; vitamin D, 3,025 IU; vitamin E, 22 mg; vitamin K3, 2.2 mg; thiamine, 1.65 mg; riboflavin, 6.6 mg; pyridoxine, 3.3 mg; cobalamin, 17.6 μg; nicotinic acid, 22 mg; pantothenic acid, 13.2 mg; folic acid, 0.33 mg; biotin, 88 μg; choline chloride, 500 mg; iron, 48 mg; zinc, 96.6 mg; manganese, 101.76 mg; copper, 10 mg; selenium, 0.05 mg; iodine, 0.96 mg; cobalt, 0.3 mg. 3Calculated value; SID means standardized ileal digestible. 4Analysed value. View Large Table 2. Composition and nutrient levels of experimental diets (%, as-fed basis). Item  Phase 1 (d 1 to 21)  Phase 2 (d 22 to 42)    CON1  1MA1  2MA1  CON  1MA  2MA  Corn  58.70  58.70  58.70  65.40  65.40  65.40  Soybean meal  30.11  30.11  30.11  22.69  22.69  22.69  Corn gluten meal  4.00  4.00  4.00  5.10  5.10  5.10  Soybean oil  3.00  2.00  1.00  3.00  2.00  1.00  Microalgae  -  1.00  2.00  -  1.00  2.00  Dicalcium phosphate  1.70  1.70  1.70  1.25  1.25  1.25  Limestone  1.39  1.39  1.39  1.44  1.44  1.44  Salt  0.30  0.30  0.30  0.30  0.30  0.30  L-lysine HCl, 78%  0.12  0.12  0.12  0.21  0.21  0.21  DL-Methionine, 98%  0.15  0.15  0.15  0.05  0.05  0.05  L-Threonine, 98%  0.03  0.03  0.03  0.06  0.06  0.06  Vitamin-mineral Premix2  0.50  0.50  0.50  0.50  0.50  0.50  Nutrient composition              Metabolic Energy, MJ/kg3  12.76  12.69  12.61  13.18  13.12  13.04  Crude Protein4  20.70  20.93  20.46  20.10  19.88  19.86  Calcium3  1.00  1.00  1.00  0.90  0.90  0.90  Available Phosphorus3  0.45  0.45  0.45  0.35  0.35  0.35  SID lysine3  1.10  1.10  1.10  1.00  1.00  1.00  SID methionine3  0.50  0.50  0.50  0.38  0.38  0.38  SID threonine3  0.80  0.80  0.80  0.74  0.74  0.74  SID tryptophan3  0.27  0.27  0.27  0.23  0.23  0.23  Item  Phase 1 (d 1 to 21)  Phase 2 (d 22 to 42)    CON1  1MA1  2MA1  CON  1MA  2MA  Corn  58.70  58.70  58.70  65.40  65.40  65.40  Soybean meal  30.11  30.11  30.11  22.69  22.69  22.69  Corn gluten meal  4.00  4.00  4.00  5.10  5.10  5.10  Soybean oil  3.00  2.00  1.00  3.00  2.00  1.00  Microalgae  -  1.00  2.00  -  1.00  2.00  Dicalcium phosphate  1.70  1.70  1.70  1.25  1.25  1.25  Limestone  1.39  1.39  1.39  1.44  1.44  1.44  Salt  0.30  0.30  0.30  0.30  0.30  0.30  L-lysine HCl, 78%  0.12  0.12  0.12  0.21  0.21  0.21  DL-Methionine, 98%  0.15  0.15  0.15  0.05  0.05  0.05  L-Threonine, 98%  0.03  0.03  0.03  0.06  0.06  0.06  Vitamin-mineral Premix2  0.50  0.50  0.50  0.50  0.50  0.50  Nutrient composition              Metabolic Energy, MJ/kg3  12.76  12.69  12.61  13.18  13.12  13.04  Crude Protein4  20.70  20.93  20.46  20.10  19.88  19.86  Calcium3  1.00  1.00  1.00  0.90  0.90  0.90  Available Phosphorus3  0.45  0.45  0.45  0.35  0.35  0.35  SID lysine3  1.10  1.10  1.10  1.00  1.00  1.00  SID methionine3  0.50  0.50  0.50  0.38  0.38  0.38  SID threonine3  0.80  0.80  0.80  0.74  0.74  0.74  SID tryptophan3  0.27  0.27  0.27  0.23  0.23  0.23  1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). 2Premix supplied per kg diet: vitamin A, 11,000 IU; vitamin D, 3,025 IU; vitamin E, 22 mg; vitamin K3, 2.2 mg; thiamine, 1.65 mg; riboflavin, 6.6 mg; pyridoxine, 3.3 mg; cobalamin, 17.6 μg; nicotinic acid, 22 mg; pantothenic acid, 13.2 mg; folic acid, 0.33 mg; biotin, 88 μg; choline chloride, 500 mg; iron, 48 mg; zinc, 96.6 mg; manganese, 101.76 mg; copper, 10 mg; selenium, 0.05 mg; iodine, 0.96 mg; cobalt, 0.3 mg. 3Calculated value; SID means standardized ileal digestible. 4Analysed value. View Large Sampling and Processing Diets were ground to pass through a 1-mm sieve before analysis. Diets were analyzed for concentrations of dry matter (DM; Method 934.01) and crude protein (CP; Method 990.03) according to the procedures of the Association of Official Analytical Chemists (AOAC, 2005), and gross energy was determined by an automatic isoperibolic oxygen bomb calorimeter (Parr 1281, Automatic Energy Analyzer; Moline, IL). On d 21 and 42, broilers were fasted for 12 h and the birds and feeders were then weighed to determine average daily gain (ADG), average daily feed intake (ADFI), and feed conversation ratio (FCR). One bird (closest to the average body weight for each pen) was euthanized for blood samples (n = 7). Blood (5 mL) was collected by cardiac puncture into a 10-mL anticoagulant-free Vacutainer tube (Greiner Bio-One GmbH, Kremsmunster, Austria) and then centrifuged at 3,000 × g for 10 min at 4°C to obtain serum. The serum samples were stored at −80°C until analysis. Measurement of Serum Indices Concentrations of glucose, albumin, globulin, albumin/globulin ratio, total protein (TP), urea nitrogen (UN), triglyceride, TC, LDL-C, high-density lipoprotein cholesterol (HDL-C) in serum samples were analyzed by an automatic biochemical analyzer (RA-1000, Bayer Corp., Tarrytown, NY) using colorimetric methods, following the instructions of the manufacturer of the corresponding reagent kit (Zhongsheng Biochemical Co., Ltd., Beijing, China). Determination of serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) levels were conducted by spectrophotometric methods using a spectrophotometer (Leng Guang SFZ1606017568, Shanghai, China) following the instructions of the kit's manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Determination of Carcass Traits The birds (n = 7, one bird closed to the average body weight from each pen) that were euthanized on d 42 were also used to determine carcass traits. The breast and thigh muscle located on the left side were removed and weighed. The percentage of the breast and thigh muscle weight relative to slaughter weight was determined. Meat color, including lightness (L*), redness (a*), and yellowness (b*) values, was measured from 3 orientations (middle, medial, and lateral) using a Chromameter (CR-410, Konica Minota, Tokyo, Japan). The pH values at 45 min and 24 h postmortem were also measured at 3 locations using a glass penetration pH electrode (pH-star, Matthaus, Germany). Drip loss for 24 h postmortem was measured using the plastic bag method as described previously (Straadt et al., 2007). On d 42, the organs of these broilers, including heart, liver, spleen, pancreas, abdominal fat, and fabricius, were collected and weighed to determine the organ percentages (organ percentage = organ weight/terminal body weight × 100%). Determination of Fatty Acid Composition At the end of d 42, milled (1-mm screen) feed (in duplicate) or homogenized skinless breast and thigh meat tissue (7 samples of each tissue from each diet) were defrosted and samples of feed (10 g), breast and thigh meat (ca. 20 g) were lyophilized for 60 h using a freeze dryer. Fatty acid profiles of the lipid sources were determined by gas chromatography (6890 series, Agilent Technologies, Wilmington, DE) according to the procedures of Sukhija and Palmquist (1988) with slight modifications. Lipid samples were converted to fatty acid methyl esters using methanolic HCl. Undecanoic acid (C11:0) was used as the internal standard. Aliquots of 1 liter were injected into a capillary column (60 m × 250 m × 250 nm, DB-23, Agilent) with cyanopropyl methyl silicone as the stationary phase. Column oven temperature was programmed with a 1:20 split. Injector and detector temperatures were maintained at 260 and 270°C, respectively. Nitrogen was the carrier gas at a flow rate of 2 mL/min. Fatty acid concentrations were then calculated in terms of milligrams of fatty acid per 100 g of feed or tissue (fresh weight). Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), n-6, n-3, and polyunsaturated fatty acids/saturated fatty acids ratio (P/S) were calculated using the following formulae:   \begin{equation*} {\rm{SFA}} = {\rm{C}}14:0 + {\rm{C}}16:0 + {\rm{C}}17:0 + {\rm{C}}18:0 + {\rm{C}}20:0 \end{equation*}   \begin{equation*} {\rm{MUFA}} = {\rm{C}}16:1{\rm{n}} - 7 + {\rm{C}}18:1{\rm{n}} - 9 \end{equation*}   \begin{eqnarray*} &&{\rm{PUFA}} = {\rm{C}}18:2{\rm{n}} - 6 + {\rm{C}}18:3{\rm{n}} - 6 + {\rm{C}}20\\ &&:4{\rm{n}} - 6 + {\rm{C}}18:3{\rm{n}} - 3 + {\rm{C}}20:5{\rm{n}} - 3 + {\rm{C}}22:6{\rm{n}} - 3 \end{eqnarray*}   \begin{equation*} {\rm{n}} - 6 = {\rm{C}}18:2{\rm{n}} - 6 + {\rm{C}}18:3{\rm{n}} - 6 + {\rm{C}}20:4{\rm{n}} - 6 \end{equation*}   \begin{equation*} {\rm{n}} - 3 = {\rm{C}}18:3{\rm{n}} - 3 + {\rm{C}}20:5{\rm{n}} - 3 + {\rm{C}}22:6{\rm{n}} - 3 \end{equation*}   \begin{equation*} {\rm{P}}/{\rm{S}} = {\rm{PUFA}}/{\rm{SFA}} \end{equation*} Statistical Analyses Data were subjected to ANOVA using the GLM procedure of SAS (SAS Institute, 1996). Pen was the experimental unit. Differences among treatments were separated by Duncan's multiple range test. Results were expressed as least squares means and SEM. Significance was designated at P ≤ 0.05, while a tendency for significance was designated at 0.05 < P ≤ 0.10. RESULTS Fatty Acid of Diets Fatty acid content of diets is shown in Table 3. The main fatty acids present in diets were C16:0, C18:0, C18:1n-9, C18:2n-6, C20:0. As MA increased, concentrations of DHA and n-3 PUFA had an increase of 2% to 3% per 1% increase of MA, while the n-6/n-3 PUFA ratio was decreased by a large margin. Table 3. Concentrations of fatty acids of diets in the experimental diets (% of total fat). Fatty acid  Phase 1  Phase 2    CON1  1MA1  2MA1  CON  1MA  2MA  C14:0 Myristic acid  0.09  0.06  1.15  0.08  0.07  0.62  C16:0 Palmitic acid  17.5  18.9  23.7  16.7  19.9  24.6  C16:1n-7 Palmitoleic acid  0.09  0.04  0.05  0.03  0.04  0.05  C17:0 Heptadecanoic acid  0.13  0.14  0.19  0.13  0.16  0.22  C18:0 Stearic acid  4.38  3.24  2.80  4.11  3.26  2.38  C18:1n-9 Oleic acid  26.3  21.5  19.2  24.2  20.4  17.3  C18:2n-6 Linoleic acid  42.2  46.1  41.0  46.0  46.2  42.1  C18:3n-6 Methyl linolenate  0.00  0.00  0.00  0.00  0.00  0.00  C18:3n-3 α-Linolenic acid  0.12  0.05  0.08  0.10  0.08  0.09  C20:0 Arachidic acid  3.18  3.91  3.09  3.33  3.57  2.30  C20:1n-9 Twitocene-enoic acid  0.53  0.41  0.38  0.51  0.43  0.40  C20:4n-6 Arachidonic acid  0.00  0.00  0.00  0.00  0.00  0.00  C20:5n-3 Eicosapentaenoic acid  0.03  0.00  0.05  0.05  0.03  0.07  C22:6n-3 Docosahexaenoic acid  0.28  2.58  5.48  0.27  2.43  6.04  Saturated fatty acids  25.2  26.2  31.0  24.4  26.9  30.1  Monounsaturated fatty acids  26.8  22.0  19.6  24.7  20.8  17.7  Polyunsaturated fatty acids  42.6  48.8  46.6  46.4  48.7  48.3  n-6 Polyunsaturated fatty acids  42.2  46.1  41.0  46.0  46.2  42.1  n-3 Polyunsaturated fatty acids  0.43  2.63  5.61  0.42  2.54  6.20  n-6/n-32  98.1  17.5  7.30  109  18.2  6.78  P/S2  1.69  1.86  1.50  1.90  1.81  1.60  Fatty acid  Phase 1  Phase 2    CON1  1MA1  2MA1  CON  1MA  2MA  C14:0 Myristic acid  0.09  0.06  1.15  0.08  0.07  0.62  C16:0 Palmitic acid  17.5  18.9  23.7  16.7  19.9  24.6  C16:1n-7 Palmitoleic acid  0.09  0.04  0.05  0.03  0.04  0.05  C17:0 Heptadecanoic acid  0.13  0.14  0.19  0.13  0.16  0.22  C18:0 Stearic acid  4.38  3.24  2.80  4.11  3.26  2.38  C18:1n-9 Oleic acid  26.3  21.5  19.2  24.2  20.4  17.3  C18:2n-6 Linoleic acid  42.2  46.1  41.0  46.0  46.2  42.1  C18:3n-6 Methyl linolenate  0.00  0.00  0.00  0.00  0.00  0.00  C18:3n-3 α-Linolenic acid  0.12  0.05  0.08  0.10  0.08  0.09  C20:0 Arachidic acid  3.18  3.91  3.09  3.33  3.57  2.30  C20:1n-9 Twitocene-enoic acid  0.53  0.41  0.38  0.51  0.43  0.40  C20:4n-6 Arachidonic acid  0.00  0.00  0.00  0.00  0.00  0.00  C20:5n-3 Eicosapentaenoic acid  0.03  0.00  0.05  0.05  0.03  0.07  C22:6n-3 Docosahexaenoic acid  0.28  2.58  5.48  0.27  2.43  6.04  Saturated fatty acids  25.2  26.2  31.0  24.4  26.9  30.1  Monounsaturated fatty acids  26.8  22.0  19.6  24.7  20.8  17.7  Polyunsaturated fatty acids  42.6  48.8  46.6  46.4  48.7  48.3  n-6 Polyunsaturated fatty acids  42.2  46.1  41.0  46.0  46.2  42.1  n-3 Polyunsaturated fatty acids  0.43  2.63  5.61  0.42  2.54  6.20  n-6/n-32  98.1  17.5  7.30  109  18.2  6.78  P/S2  1.69  1.86  1.50  1.90  1.81  1.60  1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Table 3. Concentrations of fatty acids of diets in the experimental diets (% of total fat). Fatty acid  Phase 1  Phase 2    CON1  1MA1  2MA1  CON  1MA  2MA  C14:0 Myristic acid  0.09  0.06  1.15  0.08  0.07  0.62  C16:0 Palmitic acid  17.5  18.9  23.7  16.7  19.9  24.6  C16:1n-7 Palmitoleic acid  0.09  0.04  0.05  0.03  0.04  0.05  C17:0 Heptadecanoic acid  0.13  0.14  0.19  0.13  0.16  0.22  C18:0 Stearic acid  4.38  3.24  2.80  4.11  3.26  2.38  C18:1n-9 Oleic acid  26.3  21.5  19.2  24.2  20.4  17.3  C18:2n-6 Linoleic acid  42.2  46.1  41.0  46.0  46.2  42.1  C18:3n-6 Methyl linolenate  0.00  0.00  0.00  0.00  0.00  0.00  C18:3n-3 α-Linolenic acid  0.12  0.05  0.08  0.10  0.08  0.09  C20:0 Arachidic acid  3.18  3.91  3.09  3.33  3.57  2.30  C20:1n-9 Twitocene-enoic acid  0.53  0.41  0.38  0.51  0.43  0.40  C20:4n-6 Arachidonic acid  0.00  0.00  0.00  0.00  0.00  0.00  C20:5n-3 Eicosapentaenoic acid  0.03  0.00  0.05  0.05  0.03  0.07  C22:6n-3 Docosahexaenoic acid  0.28  2.58  5.48  0.27  2.43  6.04  Saturated fatty acids  25.2  26.2  31.0  24.4  26.9  30.1  Monounsaturated fatty acids  26.8  22.0  19.6  24.7  20.8  17.7  Polyunsaturated fatty acids  42.6  48.8  46.6  46.4  48.7  48.3  n-6 Polyunsaturated fatty acids  42.2  46.1  41.0  46.0  46.2  42.1  n-3 Polyunsaturated fatty acids  0.43  2.63  5.61  0.42  2.54  6.20  n-6/n-32  98.1  17.5  7.30  109  18.2  6.78  P/S2  1.69  1.86  1.50  1.90  1.81  1.60  Fatty acid  Phase 1  Phase 2    CON1  1MA1  2MA1  CON  1MA  2MA  C14:0 Myristic acid  0.09  0.06  1.15  0.08  0.07  0.62  C16:0 Palmitic acid  17.5  18.9  23.7  16.7  19.9  24.6  C16:1n-7 Palmitoleic acid  0.09  0.04  0.05  0.03  0.04  0.05  C17:0 Heptadecanoic acid  0.13  0.14  0.19  0.13  0.16  0.22  C18:0 Stearic acid  4.38  3.24  2.80  4.11  3.26  2.38  C18:1n-9 Oleic acid  26.3  21.5  19.2  24.2  20.4  17.3  C18:2n-6 Linoleic acid  42.2  46.1  41.0  46.0  46.2  42.1  C18:3n-6 Methyl linolenate  0.00  0.00  0.00  0.00  0.00  0.00  C18:3n-3 α-Linolenic acid  0.12  0.05  0.08  0.10  0.08  0.09  C20:0 Arachidic acid  3.18  3.91  3.09  3.33  3.57  2.30  C20:1n-9 Twitocene-enoic acid  0.53  0.41  0.38  0.51  0.43  0.40  C20:4n-6 Arachidonic acid  0.00  0.00  0.00  0.00  0.00  0.00  C20:5n-3 Eicosapentaenoic acid  0.03  0.00  0.05  0.05  0.03  0.07  C22:6n-3 Docosahexaenoic acid  0.28  2.58  5.48  0.27  2.43  6.04  Saturated fatty acids  25.2  26.2  31.0  24.4  26.9  30.1  Monounsaturated fatty acids  26.8  22.0  19.6  24.7  20.8  17.7  Polyunsaturated fatty acids  42.6  48.8  46.6  46.4  48.7  48.3  n-6 Polyunsaturated fatty acids  42.2  46.1  41.0  46.0  46.2  42.1  n-3 Polyunsaturated fatty acids  0.43  2.63  5.61  0.42  2.54  6.20  n-6/n-32  98.1  17.5  7.30  109  18.2  6.78  P/S2  1.69  1.86  1.50  1.90  1.81  1.60  1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Bird Performance Performance of birds is summarized in Table 4. Compared with CON, birds supplemented with 1MA had greater (P < 0.01) body weight (BW) on d 21 and d 42, ADG in phase 1, 2 and overall (d 1 to 42), as well as higher (P < 0.01) ADFI over the 42 d experiment. Birds fed with 2MA showed higher (P < 0.01) ADG in phase 1, whereas they had lower (P < 0.01) ADFI in phase 2 and over the 42 d experiment compared with CON. Birds supplemented with MA (1MA or 2MA) had lower (P < 0.05) FCR in phase 1 and overall, while birds fed with 2MA had lower (P < 0.01) FCR in phase 2 compared with CON. Table 4. Effects of DHA-rich microalgae on performance of broilers. Item  CON1  1MA  2MA  SEM  P-value   d 0 body weight, g  44.9  46.0  45.0  0.72  0.54   d 21 body weight, g  652b  729a  707a  12.1  < 0.01   d 42 body weight, g  2,380b  2,632a  2,445b  32.5  < 0.01  d 1 to 21   Average daily gain, g/d  28.9b  32.5a  31.5a  0.57  < 0.01   Average daily feed intake, g/d  43.3  45.0  43.5  0.95  0.41   Feed conversation ratio, g/g  1.50a  1.39b  1.38b  0.03  0.02  d 22 to 42   Average daily gain, g/d  82.3b  90.7a  82.8b  1.40  < 0.01   Average daily feed intake, g/d  139a  149a  124b  3.33  < 0.01   Feed conversation ratio, g/g  1.70a  1.65a  1.50b  0.04  < 0.01  d 1 to 42   Average daily gain, g/d  55.6b  61.6a  57.1b  0.78  < 0.01   Average daily feed intake, g/d  91.3b  97.1a  83.7c  1.70  < 0.01   Feed conversation ratio, g/g  1.60a  1.52b  1.44b  0.03  < 0.01  Item  CON1  1MA  2MA  SEM  P-value   d 0 body weight, g  44.9  46.0  45.0  0.72  0.54   d 21 body weight, g  652b  729a  707a  12.1  < 0.01   d 42 body weight, g  2,380b  2,632a  2,445b  32.5  < 0.01  d 1 to 21   Average daily gain, g/d  28.9b  32.5a  31.5a  0.57  < 0.01   Average daily feed intake, g/d  43.3  45.0  43.5  0.95  0.41   Feed conversation ratio, g/g  1.50a  1.39b  1.38b  0.03  0.02  d 22 to 42   Average daily gain, g/d  82.3b  90.7a  82.8b  1.40  < 0.01   Average daily feed intake, g/d  139a  149a  124b  3.33  < 0.01   Feed conversation ratio, g/g  1.70a  1.65a  1.50b  0.04  < 0.01  d 1 to 42   Average daily gain, g/d  55.6b  61.6a  57.1b  0.78  < 0.01   Average daily feed intake, g/d  91.3b  97.1a  83.7c  1.70  < 0.01   Feed conversation ratio, g/g  1.60a  1.52b  1.44b  0.03  < 0.01  SEM means standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). View Large Table 4. Effects of DHA-rich microalgae on performance of broilers. Item  CON1  1MA  2MA  SEM  P-value   d 0 body weight, g  44.9  46.0  45.0  0.72  0.54   d 21 body weight, g  652b  729a  707a  12.1  < 0.01   d 42 body weight, g  2,380b  2,632a  2,445b  32.5  < 0.01  d 1 to 21   Average daily gain, g/d  28.9b  32.5a  31.5a  0.57  < 0.01   Average daily feed intake, g/d  43.3  45.0  43.5  0.95  0.41   Feed conversation ratio, g/g  1.50a  1.39b  1.38b  0.03  0.02  d 22 to 42   Average daily gain, g/d  82.3b  90.7a  82.8b  1.40  < 0.01   Average daily feed intake, g/d  139a  149a  124b  3.33  < 0.01   Feed conversation ratio, g/g  1.70a  1.65a  1.50b  0.04  < 0.01  d 1 to 42   Average daily gain, g/d  55.6b  61.6a  57.1b  0.78  < 0.01   Average daily feed intake, g/d  91.3b  97.1a  83.7c  1.70  < 0.01   Feed conversation ratio, g/g  1.60a  1.52b  1.44b  0.03  < 0.01  Item  CON1  1MA  2MA  SEM  P-value   d 0 body weight, g  44.9  46.0  45.0  0.72  0.54   d 21 body weight, g  652b  729a  707a  12.1  < 0.01   d 42 body weight, g  2,380b  2,632a  2,445b  32.5  < 0.01  d 1 to 21   Average daily gain, g/d  28.9b  32.5a  31.5a  0.57  < 0.01   Average daily feed intake, g/d  43.3  45.0  43.5  0.95  0.41   Feed conversation ratio, g/g  1.50a  1.39b  1.38b  0.03  0.02  d 22 to 42   Average daily gain, g/d  82.3b  90.7a  82.8b  1.40  < 0.01   Average daily feed intake, g/d  139a  149a  124b  3.33  < 0.01   Feed conversation ratio, g/g  1.70a  1.65a  1.50b  0.04  < 0.01  d 1 to 42   Average daily gain, g/d  55.6b  61.6a  57.1b  0.78  < 0.01   Average daily feed intake, g/d  91.3b  97.1a  83.7c  1.70  < 0.01   Feed conversation ratio, g/g  1.60a  1.52b  1.44b  0.03  < 0.01  SEM means standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). View Large Carcass Traits Effects of MA on carcass traits of birds are shown in Table 5. Breast muscle yield was lower (P < 0.05) in birds fed with 2MA compared with those supplemented with 1MA. Thigh muscle yield tended to be higher (P = 0.10) in birds supplemented with 1MA compared with CON. There were no negative effects of MA supplementation on thigh and breast pH value, lightness, redness, yellowness, or drip loss of birds, while MA increased (P < 0.05) liver percentage (liver weight/body weight) and decreased (P < 0.05) abdominal fat percentage (abdominal fat weight/body weight) compared with CON. Birds supplemented with 2MA had higher (P < 0.05) spleen percentage (spleen weight/body weight) compared with CON. Table 5. Effects of DHA-rich microalgae on carcass traits of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle   Breast muscle yield, %  7.90a,b  8.45a  7.28b  0.26  0.03   pH40 min  6.45  6.41  6.57  0.08  0.38   pH24 h  5.26  5.33  5.44  0.08  0.28   Lightness  38.0  37.6  37.1  0.70  0.67   Redness  3.34  3.41  2.90  0.47  0.72   Yellowness  8.94  9.00  9.16  0.31  0.89   Drip loss, %  2.14  1.91  1.76  0.53  0.83  Thigh muscle   Thigh muscle yield, %  9.44d  10.2c  9.34d  0.28  0.10   pH40 min  6.46  6.40  6.36  0.07  0.67   pH24 h  5.30  5.34  5.23  0.08  0.61   Lightness  39.1  39.2  40.2  0.88  0.64   Redness  4.40  4.68  4.76  0.35  0.75   Yellowness  10.0  10.4  10.3  0.44  0.85   Drip loss, %  0.71  0.87  1.08  0.13  0.17  Slaughter weight, kg  2.51  2.59  2.43  54.2  0.16   Heart index percentage  0.58  0.71  0.67  0.07  0.42   Liver percentage  1.98b  2.56a  2.49a  0.14  0.02   Spleen percentage  1.07b  1.21b  1.78a  0.16  0.02   Pancreas percentage  0.17  0.19  0.20  0.01  0.23   Abdominal fat percentage  0.20a  0.15b  0.14b  0.02  0.04   Fabricius percentage  0.24  0.23  0.22  0.03  0.88  Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle   Breast muscle yield, %  7.90a,b  8.45a  7.28b  0.26  0.03   pH40 min  6.45  6.41  6.57  0.08  0.38   pH24 h  5.26  5.33  5.44  0.08  0.28   Lightness  38.0  37.6  37.1  0.70  0.67   Redness  3.34  3.41  2.90  0.47  0.72   Yellowness  8.94  9.00  9.16  0.31  0.89   Drip loss, %  2.14  1.91  1.76  0.53  0.83  Thigh muscle   Thigh muscle yield, %  9.44d  10.2c  9.34d  0.28  0.10   pH40 min  6.46  6.40  6.36  0.07  0.67   pH24 h  5.30  5.34  5.23  0.08  0.61   Lightness  39.1  39.2  40.2  0.88  0.64   Redness  4.40  4.68  4.76  0.35  0.75   Yellowness  10.0  10.4  10.3  0.44  0.85   Drip loss, %  0.71  0.87  1.08  0.13  0.17  Slaughter weight, kg  2.51  2.59  2.43  54.2  0.16   Heart index percentage  0.58  0.71  0.67  0.07  0.42   Liver percentage  1.98b  2.56a  2.49a  0.14  0.02   Spleen percentage  1.07b  1.21b  1.78a  0.16  0.02   Pancreas percentage  0.17  0.19  0.20  0.01  0.23   Abdominal fat percentage  0.20a  0.15b  0.14b  0.02  0.04   Fabricius percentage  0.24  0.23  0.22  0.03  0.88  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). View Large Table 5. Effects of DHA-rich microalgae on carcass traits of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle   Breast muscle yield, %  7.90a,b  8.45a  7.28b  0.26  0.03   pH40 min  6.45  6.41  6.57  0.08  0.38   pH24 h  5.26  5.33  5.44  0.08  0.28   Lightness  38.0  37.6  37.1  0.70  0.67   Redness  3.34  3.41  2.90  0.47  0.72   Yellowness  8.94  9.00  9.16  0.31  0.89   Drip loss, %  2.14  1.91  1.76  0.53  0.83  Thigh muscle   Thigh muscle yield, %  9.44d  10.2c  9.34d  0.28  0.10   pH40 min  6.46  6.40  6.36  0.07  0.67   pH24 h  5.30  5.34  5.23  0.08  0.61   Lightness  39.1  39.2  40.2  0.88  0.64   Redness  4.40  4.68  4.76  0.35  0.75   Yellowness  10.0  10.4  10.3  0.44  0.85   Drip loss, %  0.71  0.87  1.08  0.13  0.17  Slaughter weight, kg  2.51  2.59  2.43  54.2  0.16   Heart index percentage  0.58  0.71  0.67  0.07  0.42   Liver percentage  1.98b  2.56a  2.49a  0.14  0.02   Spleen percentage  1.07b  1.21b  1.78a  0.16  0.02   Pancreas percentage  0.17  0.19  0.20  0.01  0.23   Abdominal fat percentage  0.20a  0.15b  0.14b  0.02  0.04   Fabricius percentage  0.24  0.23  0.22  0.03  0.88  Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle   Breast muscle yield, %  7.90a,b  8.45a  7.28b  0.26  0.03   pH40 min  6.45  6.41  6.57  0.08  0.38   pH24 h  5.26  5.33  5.44  0.08  0.28   Lightness  38.0  37.6  37.1  0.70  0.67   Redness  3.34  3.41  2.90  0.47  0.72   Yellowness  8.94  9.00  9.16  0.31  0.89   Drip loss, %  2.14  1.91  1.76  0.53  0.83  Thigh muscle   Thigh muscle yield, %  9.44d  10.2c  9.34d  0.28  0.10   pH40 min  6.46  6.40  6.36  0.07  0.67   pH24 h  5.30  5.34  5.23  0.08  0.61   Lightness  39.1  39.2  40.2  0.88  0.64   Redness  4.40  4.68  4.76  0.35  0.75   Yellowness  10.0  10.4  10.3  0.44  0.85   Drip loss, %  0.71  0.87  1.08  0.13  0.17  Slaughter weight, kg  2.51  2.59  2.43  54.2  0.16   Heart index percentage  0.58  0.71  0.67  0.07  0.42   Liver percentage  1.98b  2.56a  2.49a  0.14  0.02   Spleen percentage  1.07b  1.21b  1.78a  0.16  0.02   Pancreas percentage  0.17  0.19  0.20  0.01  0.23   Abdominal fat percentage  0.20a  0.15b  0.14b  0.02  0.04   Fabricius percentage  0.24  0.23  0.22  0.03  0.88  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil (hereinafter the same). View Large Serological Index Effects of MA on serological index of birds are presented in Table 6. In phase 1, birds fed 2MA showed higher (P < 0.05) levels of serum glucose and albumin/globulin ratio, while birds supplemented with MA had lower (P ≤ 0.05) concentration of TC and LDL-C in serum compared with CON. In phase 2, birds supplemented with MA had higher (P < 0.01) concentration of glucose and a trend to increase (P = 0.09) TP, while birds fed with MA also showed lower (P < 0.01) concentration of serum UN and tended to decrease (P = 0.08) concentration of TC in serum compared with CON. Concentration of LDL-C in serum tended to be lower (P = 0.07) in birds supplemented with 1MA, whereas content of HDL-C in serum tended to increase (P = 0.09) in birds fed with 2MA in phase 2 compared with CON. Table 6. Effects of DHA-rich microalgae on serological index of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  d 1 to 21   Glucose, mmol/L  17.6b  18.7a,b  19.6a  0.43  0.03   Albumin, g/L  11.9  12.6  12.9  0.72  0.66   Globulin, g/L  18.7  19.9  19.9  1.24  0.84   Albumin/globulin ratio  0.63b  0.63b  0.66a  0.01  0.02   Total protein, g/L  30.6  32.5  32.8  1.95  0.99   Urea nitrogen, mmol/L  0.48  0.50  0.44  0.04  0.95   Triglycerides, mmol/L  0.99  0.43  0.76  0.14  0.22   Total cholesterol, mmol/L  4.57a  3.62b  3.42b  0.21  0.05   LDL-C, mmol/L2  0.52a  0.38b  0.43b  0.03  0.05   HDL-C, mmol/L2  1.53  1.62  1.61  0.07  0.56  d 22 to 42   Glucose, mmol/L  14.5b  18.6a  16.9a  0.63  < 0.01   Albumin, g/L  11.6  12.8  12.6  0.39  0.22   Globulin, g/L  16.2  22.0  20.8  1.75  0.14   Albumin/globulin ratio  0.73  0.60  0.61  0.05  0.23   Total protein, g/L  27.8d  34.8c  33.4c  1.95  0.09   Urea nitrogen, mmol/L  0.40a  0.23b  0.34a  0.02  < 0.01   Triglycerides, mmol/L  0.77  0.70  0.50  0.09  0.46   Total cholesterol, mmol/L  3.30c  2.73d  2.70d  0.16  0.08   LDL-C, mmol/L  0.57c  0.34d  0.42c,d  0.05  0.07   HDL-C, mmol/L  1.13d  1.25c,d  1.40c  0.09  0.09  Item  CON1  1MA1  2MA1  SEM  P-value  d 1 to 21   Glucose, mmol/L  17.6b  18.7a,b  19.6a  0.43  0.03   Albumin, g/L  11.9  12.6  12.9  0.72  0.66   Globulin, g/L  18.7  19.9  19.9  1.24  0.84   Albumin/globulin ratio  0.63b  0.63b  0.66a  0.01  0.02   Total protein, g/L  30.6  32.5  32.8  1.95  0.99   Urea nitrogen, mmol/L  0.48  0.50  0.44  0.04  0.95   Triglycerides, mmol/L  0.99  0.43  0.76  0.14  0.22   Total cholesterol, mmol/L  4.57a  3.62b  3.42b  0.21  0.05   LDL-C, mmol/L2  0.52a  0.38b  0.43b  0.03  0.05   HDL-C, mmol/L2  1.53  1.62  1.61  0.07  0.56  d 22 to 42   Glucose, mmol/L  14.5b  18.6a  16.9a  0.63  < 0.01   Albumin, g/L  11.6  12.8  12.6  0.39  0.22   Globulin, g/L  16.2  22.0  20.8  1.75  0.14   Albumin/globulin ratio  0.73  0.60  0.61  0.05  0.23   Total protein, g/L  27.8d  34.8c  33.4c  1.95  0.09   Urea nitrogen, mmol/L  0.40a  0.23b  0.34a  0.02  < 0.01   Triglycerides, mmol/L  0.77  0.70  0.50  0.09  0.46   Total cholesterol, mmol/L  3.30c  2.73d  2.70d  0.16  0.08   LDL-C, mmol/L  0.57c  0.34d  0.42c,d  0.05  0.07   HDL-C, mmol/L  1.13d  1.25c,d  1.40c  0.09  0.09  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol. View Large Table 6. Effects of DHA-rich microalgae on serological index of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  d 1 to 21   Glucose, mmol/L  17.6b  18.7a,b  19.6a  0.43  0.03   Albumin, g/L  11.9  12.6  12.9  0.72  0.66   Globulin, g/L  18.7  19.9  19.9  1.24  0.84   Albumin/globulin ratio  0.63b  0.63b  0.66a  0.01  0.02   Total protein, g/L  30.6  32.5  32.8  1.95  0.99   Urea nitrogen, mmol/L  0.48  0.50  0.44  0.04  0.95   Triglycerides, mmol/L  0.99  0.43  0.76  0.14  0.22   Total cholesterol, mmol/L  4.57a  3.62b  3.42b  0.21  0.05   LDL-C, mmol/L2  0.52a  0.38b  0.43b  0.03  0.05   HDL-C, mmol/L2  1.53  1.62  1.61  0.07  0.56  d 22 to 42   Glucose, mmol/L  14.5b  18.6a  16.9a  0.63  < 0.01   Albumin, g/L  11.6  12.8  12.6  0.39  0.22   Globulin, g/L  16.2  22.0  20.8  1.75  0.14   Albumin/globulin ratio  0.73  0.60  0.61  0.05  0.23   Total protein, g/L  27.8d  34.8c  33.4c  1.95  0.09   Urea nitrogen, mmol/L  0.40a  0.23b  0.34a  0.02  < 0.01   Triglycerides, mmol/L  0.77  0.70  0.50  0.09  0.46   Total cholesterol, mmol/L  3.30c  2.73d  2.70d  0.16  0.08   LDL-C, mmol/L  0.57c  0.34d  0.42c,d  0.05  0.07   HDL-C, mmol/L  1.13d  1.25c,d  1.40c  0.09  0.09  Item  CON1  1MA1  2MA1  SEM  P-value  d 1 to 21   Glucose, mmol/L  17.6b  18.7a,b  19.6a  0.43  0.03   Albumin, g/L  11.9  12.6  12.9  0.72  0.66   Globulin, g/L  18.7  19.9  19.9  1.24  0.84   Albumin/globulin ratio  0.63b  0.63b  0.66a  0.01  0.02   Total protein, g/L  30.6  32.5  32.8  1.95  0.99   Urea nitrogen, mmol/L  0.48  0.50  0.44  0.04  0.95   Triglycerides, mmol/L  0.99  0.43  0.76  0.14  0.22   Total cholesterol, mmol/L  4.57a  3.62b  3.42b  0.21  0.05   LDL-C, mmol/L2  0.52a  0.38b  0.43b  0.03  0.05   HDL-C, mmol/L2  1.53  1.62  1.61  0.07  0.56  d 22 to 42   Glucose, mmol/L  14.5b  18.6a  16.9a  0.63  < 0.01   Albumin, g/L  11.6  12.8  12.6  0.39  0.22   Globulin, g/L  16.2  22.0  20.8  1.75  0.14   Albumin/globulin ratio  0.73  0.60  0.61  0.05  0.23   Total protein, g/L  27.8d  34.8c  33.4c  1.95  0.09   Urea nitrogen, mmol/L  0.40a  0.23b  0.34a  0.02  < 0.01   Triglycerides, mmol/L  0.77  0.70  0.50  0.09  0.46   Total cholesterol, mmol/L  3.30c  2.73d  2.70d  0.16  0.08   LDL-C, mmol/L  0.57c  0.34d  0.42c,d  0.05  0.07   HDL-C, mmol/L  1.13d  1.25c,d  1.40c  0.09  0.09  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol. View Large Fatty Acid in Meat Effects of MA on fatty acid content in breast and thigh muscle for birds are shown in Tables 7 and 8. In breast muscle (Table 7), birds fed with MA had increased (P < 0.05) concentration of C14:0, C17:0, C20:5n-3, C22:6n-3, SFA, and n-3 PUFA, and decreased (P < 0.01) content of C18:2n-6, n-6 PUFA, n-6/n-3 PUFA ratio, and P/S compared with CON. Meanwhile, birds fed with 2MA showed higher (P < 0.05) concentration of C16:0, whereas lower (P < 0.05) content of C18:3n-3 and a trend for decreased (P = 0.06) concentration of C18:3n-6 in breast muscle compared with CON. In thigh muscle (Table 8), birds fed with MA had increased (P < 0.05) concentration of C14:0, C20:5n-3, C22:6n-3, and n-3 PUFA, and decreased (P < 0.01) content of C18:2n-6, C18:3n-6, C18:3n-3, n-6/n-3 PUFA ratio, and P/S compared with CON. Meanwhile, birds fed with 2MA showed lower (P < 0.05) content of n-6 PUFA, while birds supplemented with 1MA had decreased (P < 0.05) concentration of C20:0 in thigh muscle compared with CON. Table 7. Effects of DHA-rich microalgae on fatty acid of breast muscle for broilers (g/100 g fresh muscle). Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.47b  0.63a  0.74a  0.04  0.01  C16:0 Palmitic acid  21.8b  23.7a,b  24.9a  0.59  0.03  C16:1n-7 Palmitoleic acid  4.18  4.13  4.67  0.26  0.12  C17:0 Heptadecanoic acid  0.12b  0.15a  0.16a  0.01  0.02  C18:0 Stearic acid  8.40  8.29  8.70  0.56  0.99  C18:1n-9 Oleic acid  31.9  30.5  29.1  1.13  0.34  C18:2n-6 Linoleic acid  25.6a  22.5b  18.6c  0.51  < 0.01  C18:3n-6 Methyl linolenate  0.28d  0.22d,e  0.18e  0.02  0.06  C18:3n-3 α-Linolenic acid  2.02a  1.72a  1.19b  0.12  0.01  C20:0 Arachidic acid  0.12  0.10  0.12  0.01  0.33  C20:1n-9 Twitocene-enoic acid  0.25  0.28  0.27  0.02  0.70  C20:4n-6 Arachidonic acid  2.46  2.44  2.50  0.56  1.00  C20:5n-3 Eicosapentaenoic acid  0.12c  0.34b  0.61a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.30c  2.75b  5.71a  0.62  < 0.01  Saturated fatty acids  30.9c  32.8b  34.6a  0.44  < 0.01  Polyunsaturated fatty acids  30.8  29.9  28.8  0.79  0.17  n-6 Polyunsaturated fatty acids  28.4a  25.1b  21.3c  0.62  < 0.01  n-3 Polyunsaturated fatty acids  2.44c  4.81b  7.52a  0.53  < 0.01  n-6/n-32  11.7a  5.22b  2.94c  0.29  < 0.01  P/S2  1.00a  0.91b  0.83c  0.02  < 0.01  Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.47b  0.63a  0.74a  0.04  0.01  C16:0 Palmitic acid  21.8b  23.7a,b  24.9a  0.59  0.03  C16:1n-7 Palmitoleic acid  4.18  4.13  4.67  0.26  0.12  C17:0 Heptadecanoic acid  0.12b  0.15a  0.16a  0.01  0.02  C18:0 Stearic acid  8.40  8.29  8.70  0.56  0.99  C18:1n-9 Oleic acid  31.9  30.5  29.1  1.13  0.34  C18:2n-6 Linoleic acid  25.6a  22.5b  18.6c  0.51  < 0.01  C18:3n-6 Methyl linolenate  0.28d  0.22d,e  0.18e  0.02  0.06  C18:3n-3 α-Linolenic acid  2.02a  1.72a  1.19b  0.12  0.01  C20:0 Arachidic acid  0.12  0.10  0.12  0.01  0.33  C20:1n-9 Twitocene-enoic acid  0.25  0.28  0.27  0.02  0.70  C20:4n-6 Arachidonic acid  2.46  2.44  2.50  0.56  1.00  C20:5n-3 Eicosapentaenoic acid  0.12c  0.34b  0.61a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.30c  2.75b  5.71a  0.62  < 0.01  Saturated fatty acids  30.9c  32.8b  34.6a  0.44  < 0.01  Polyunsaturated fatty acids  30.8  29.9  28.8  0.79  0.17  n-6 Polyunsaturated fatty acids  28.4a  25.1b  21.3c  0.62  < 0.01  n-3 Polyunsaturated fatty acids  2.44c  4.81b  7.52a  0.53  < 0.01  n-6/n-32  11.7a  5.22b  2.94c  0.29  < 0.01  P/S2  1.00a  0.91b  0.83c  0.02  < 0.01  SEM, standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). d-eDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Table 7. Effects of DHA-rich microalgae on fatty acid of breast muscle for broilers (g/100 g fresh muscle). Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.47b  0.63a  0.74a  0.04  0.01  C16:0 Palmitic acid  21.8b  23.7a,b  24.9a  0.59  0.03  C16:1n-7 Palmitoleic acid  4.18  4.13  4.67  0.26  0.12  C17:0 Heptadecanoic acid  0.12b  0.15a  0.16a  0.01  0.02  C18:0 Stearic acid  8.40  8.29  8.70  0.56  0.99  C18:1n-9 Oleic acid  31.9  30.5  29.1  1.13  0.34  C18:2n-6 Linoleic acid  25.6a  22.5b  18.6c  0.51  < 0.01  C18:3n-6 Methyl linolenate  0.28d  0.22d,e  0.18e  0.02  0.06  C18:3n-3 α-Linolenic acid  2.02a  1.72a  1.19b  0.12  0.01  C20:0 Arachidic acid  0.12  0.10  0.12  0.01  0.33  C20:1n-9 Twitocene-enoic acid  0.25  0.28  0.27  0.02  0.70  C20:4n-6 Arachidonic acid  2.46  2.44  2.50  0.56  1.00  C20:5n-3 Eicosapentaenoic acid  0.12c  0.34b  0.61a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.30c  2.75b  5.71a  0.62  < 0.01  Saturated fatty acids  30.9c  32.8b  34.6a  0.44  < 0.01  Polyunsaturated fatty acids  30.8  29.9  28.8  0.79  0.17  n-6 Polyunsaturated fatty acids  28.4a  25.1b  21.3c  0.62  < 0.01  n-3 Polyunsaturated fatty acids  2.44c  4.81b  7.52a  0.53  < 0.01  n-6/n-32  11.7a  5.22b  2.94c  0.29  < 0.01  P/S2  1.00a  0.91b  0.83c  0.02  < 0.01  Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.47b  0.63a  0.74a  0.04  0.01  C16:0 Palmitic acid  21.8b  23.7a,b  24.9a  0.59  0.03  C16:1n-7 Palmitoleic acid  4.18  4.13  4.67  0.26  0.12  C17:0 Heptadecanoic acid  0.12b  0.15a  0.16a  0.01  0.02  C18:0 Stearic acid  8.40  8.29  8.70  0.56  0.99  C18:1n-9 Oleic acid  31.9  30.5  29.1  1.13  0.34  C18:2n-6 Linoleic acid  25.6a  22.5b  18.6c  0.51  < 0.01  C18:3n-6 Methyl linolenate  0.28d  0.22d,e  0.18e  0.02  0.06  C18:3n-3 α-Linolenic acid  2.02a  1.72a  1.19b  0.12  0.01  C20:0 Arachidic acid  0.12  0.10  0.12  0.01  0.33  C20:1n-9 Twitocene-enoic acid  0.25  0.28  0.27  0.02  0.70  C20:4n-6 Arachidonic acid  2.46  2.44  2.50  0.56  1.00  C20:5n-3 Eicosapentaenoic acid  0.12c  0.34b  0.61a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.30c  2.75b  5.71a  0.62  < 0.01  Saturated fatty acids  30.9c  32.8b  34.6a  0.44  < 0.01  Polyunsaturated fatty acids  30.8  29.9  28.8  0.79  0.17  n-6 Polyunsaturated fatty acids  28.4a  25.1b  21.3c  0.62  < 0.01  n-3 Polyunsaturated fatty acids  2.44c  4.81b  7.52a  0.53  < 0.01  n-6/n-32  11.7a  5.22b  2.94c  0.29  < 0.01  P/S2  1.00a  0.91b  0.83c  0.02  < 0.01  SEM, standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). d-eDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Table 8. Effects of DHA-rich microalgae on fatty acid of thigh muscle for broilers (g/100 g fresh muscle). Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.49b  0.63a  0.64a  0.03  0.02  C16:0 Palmitic acid  24.4  23.4  23.3  1.70  0.86  C16:1n-7 Palmitoleic acid  4.55  3.80  3.54  0.45  0.31  C17:0 Heptadecanoic acid  0.13  0.16  0.16  0.01  0.36  C18:0 Stearic acid  9.46  8.84  9.77  0.93  0.50  C18:1n-9 Oleic acid  22.7  30.0  28.9  6.36  0.62  C18:2n-6 Linoleic acid  28.0a  21.7b  16.1b  1.74  0.01  C18:3n-6 Methyl linolenate  0.33a  0.23b  0.21b  0.02  0.04  C18:3n-3 α-Linolenic acid  2.25a  1.62b  0.93c  0.11  < 0.01  C20:0 Arachidic acid  0.18a  0.13b  0.17a  0.01  0.04  C20:1n-9 Twitocene-enoic acid  0.30  0.25  0.29  0.06  0.60  C20:4n-6 Arachidonic acid  3.51  2.93  3.09  0.78  0.79  C20:5n-3 Eicosapentaenoic acid  0.24c  0.35b  0.54a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.43c  3.44b  8.64a  0.21  < 0.01  Saturated fatty acids  34.7  33.2  34.1  2.59  0.82  Polyunsaturated fatty acids  34.8  30.2  29.5  2.69  0.35  n-6 Polyunsaturated fatty acids  31.8a  24.8a,b  19.4b  2.52  0.04  n-3 Polyunsaturated fatty acids  2.92c  5.41b  10.1a  0.27  < 0.01  n-6/n-32  10.9a  4.62b  1.92c  0.38  < 0.01  P/S2  1.00a  0.91b  0.87b  0.02  0.01  Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.49b  0.63a  0.64a  0.03  0.02  C16:0 Palmitic acid  24.4  23.4  23.3  1.70  0.86  C16:1n-7 Palmitoleic acid  4.55  3.80  3.54  0.45  0.31  C17:0 Heptadecanoic acid  0.13  0.16  0.16  0.01  0.36  C18:0 Stearic acid  9.46  8.84  9.77  0.93  0.50  C18:1n-9 Oleic acid  22.7  30.0  28.9  6.36  0.62  C18:2n-6 Linoleic acid  28.0a  21.7b  16.1b  1.74  0.01  C18:3n-6 Methyl linolenate  0.33a  0.23b  0.21b  0.02  0.04  C18:3n-3 α-Linolenic acid  2.25a  1.62b  0.93c  0.11  < 0.01  C20:0 Arachidic acid  0.18a  0.13b  0.17a  0.01  0.04  C20:1n-9 Twitocene-enoic acid  0.30  0.25  0.29  0.06  0.60  C20:4n-6 Arachidonic acid  3.51  2.93  3.09  0.78  0.79  C20:5n-3 Eicosapentaenoic acid  0.24c  0.35b  0.54a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.43c  3.44b  8.64a  0.21  < 0.01  Saturated fatty acids  34.7  33.2  34.1  2.59  0.82  Polyunsaturated fatty acids  34.8  30.2  29.5  2.69  0.35  n-6 Polyunsaturated fatty acids  31.8a  24.8a,b  19.4b  2.52  0.04  n-3 Polyunsaturated fatty acids  2.92c  5.41b  10.1a  0.27  < 0.01  n-6/n-32  10.9a  4.62b  1.92c  0.38  < 0.01  P/S2  1.00a  0.91b  0.87b  0.02  0.01  SEM, standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Table 8. Effects of DHA-rich microalgae on fatty acid of thigh muscle for broilers (g/100 g fresh muscle). Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.49b  0.63a  0.64a  0.03  0.02  C16:0 Palmitic acid  24.4  23.4  23.3  1.70  0.86  C16:1n-7 Palmitoleic acid  4.55  3.80  3.54  0.45  0.31  C17:0 Heptadecanoic acid  0.13  0.16  0.16  0.01  0.36  C18:0 Stearic acid  9.46  8.84  9.77  0.93  0.50  C18:1n-9 Oleic acid  22.7  30.0  28.9  6.36  0.62  C18:2n-6 Linoleic acid  28.0a  21.7b  16.1b  1.74  0.01  C18:3n-6 Methyl linolenate  0.33a  0.23b  0.21b  0.02  0.04  C18:3n-3 α-Linolenic acid  2.25a  1.62b  0.93c  0.11  < 0.01  C20:0 Arachidic acid  0.18a  0.13b  0.17a  0.01  0.04  C20:1n-9 Twitocene-enoic acid  0.30  0.25  0.29  0.06  0.60  C20:4n-6 Arachidonic acid  3.51  2.93  3.09  0.78  0.79  C20:5n-3 Eicosapentaenoic acid  0.24c  0.35b  0.54a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.43c  3.44b  8.64a  0.21  < 0.01  Saturated fatty acids  34.7  33.2  34.1  2.59  0.82  Polyunsaturated fatty acids  34.8  30.2  29.5  2.69  0.35  n-6 Polyunsaturated fatty acids  31.8a  24.8a,b  19.4b  2.52  0.04  n-3 Polyunsaturated fatty acids  2.92c  5.41b  10.1a  0.27  < 0.01  n-6/n-32  10.9a  4.62b  1.92c  0.38  < 0.01  P/S2  1.00a  0.91b  0.87b  0.02  0.01  Item  CON1  1MA1  2MA1  SEM  P-value  C14:0 Myristic acid  0.49b  0.63a  0.64a  0.03  0.02  C16:0 Palmitic acid  24.4  23.4  23.3  1.70  0.86  C16:1n-7 Palmitoleic acid  4.55  3.80  3.54  0.45  0.31  C17:0 Heptadecanoic acid  0.13  0.16  0.16  0.01  0.36  C18:0 Stearic acid  9.46  8.84  9.77  0.93  0.50  C18:1n-9 Oleic acid  22.7  30.0  28.9  6.36  0.62  C18:2n-6 Linoleic acid  28.0a  21.7b  16.1b  1.74  0.01  C18:3n-6 Methyl linolenate  0.33a  0.23b  0.21b  0.02  0.04  C18:3n-3 α-Linolenic acid  2.25a  1.62b  0.93c  0.11  < 0.01  C20:0 Arachidic acid  0.18a  0.13b  0.17a  0.01  0.04  C20:1n-9 Twitocene-enoic acid  0.30  0.25  0.29  0.06  0.60  C20:4n-6 Arachidonic acid  3.51  2.93  3.09  0.78  0.79  C20:5n-3 Eicosapentaenoic acid  0.24c  0.35b  0.54a  0.02  < 0.01  C22:6n-3 Docosahexaenoic acid  0.43c  3.44b  8.64a  0.21  < 0.01  Saturated fatty acids  34.7  33.2  34.1  2.59  0.82  Polyunsaturated fatty acids  34.8  30.2  29.5  2.69  0.35  n-6 Polyunsaturated fatty acids  31.8a  24.8a,b  19.4b  2.52  0.04  n-3 Polyunsaturated fatty acids  2.92c  5.41b  10.1a  0.27  < 0.01  n-6/n-32  10.9a  4.62b  1.92c  0.38  < 0.01  P/S2  1.00a  0.91b  0.87b  0.02  0.01  SEM, standard error of the mean. a-cDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. 2n-6/n-3: n-6/n-3 Polyunsaturated fatty acids ratio; P/S: Polyunsaturated fatty acids/Saturated fatty acids ratio. View Large Antioxidant Capacity Effects of MA on antioxidant capacity of broilers are shown in Table 9. In breast muscle, birds fed with 1MA showed greater (P < 0.05) concentration of SOD, CAT, and a trend of improved content of T-AOC (P = 0.07), while they also had a lower (P < 0.05) level of MDA compared with CON. Birds supplemented with 2MA had improved (P < 0.05) content of SOD compared with CON. In thigh muscle, the content of T-AOC and SOD was higher (P < 0.05) in birds fed with 1MA, while the concentration of GSH-Px was greater (P < 0.05) in birds fed with 2MA, and the content of MDA tended to be lower (P = 0.09) in birds supplemented with 1MA compared with CON. Table 9. Effects of DHA-rich microalgae on antioxidant capacity in breast and thigh muscle of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle:   Superoxide dismutase, U/mg  114b  162a  158a  10.9  0.05   Total antioxidant capacity, U/mg  5.63d  9.90c  9.01c,d  0.97  0.07   Glutathione peroxidase, U/mg  699  925  855  63.3  0.14   Catalase, U/mg  5.26b  9.78a  5.82b  0.80  0.03   Malondialdehyde, nmol/mg  5.64a  3.67b  4.35a,b  0.41  0.05  Thigh muscle:   Superoxide dismutase, U/mg  118b  185a  163a,b  16.1  0.05   Total antioxidant capacity, U/mg  5.71b  10.36a  7.61b  0.53  < 0.01   Glutathione peroxidase, U/mg  661b  853a,b  964a  55.2  0.04   Catalase, U/mg  6.36  9.25  6.15  0.91  0.13   Malondialdehyde, nmol/mg  4.67c  3.56d  4.16c,d  0.26  0.09  Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle:   Superoxide dismutase, U/mg  114b  162a  158a  10.9  0.05   Total antioxidant capacity, U/mg  5.63d  9.90c  9.01c,d  0.97  0.07   Glutathione peroxidase, U/mg  699  925  855  63.3  0.14   Catalase, U/mg  5.26b  9.78a  5.82b  0.80  0.03   Malondialdehyde, nmol/mg  5.64a  3.67b  4.35a,b  0.41  0.05  Thigh muscle:   Superoxide dismutase, U/mg  118b  185a  163a,b  16.1  0.05   Total antioxidant capacity, U/mg  5.71b  10.36a  7.61b  0.53  < 0.01   Glutathione peroxidase, U/mg  661b  853a,b  964a  55.2  0.04   Catalase, U/mg  6.36  9.25  6.15  0.91  0.13   Malondialdehyde, nmol/mg  4.67c  3.56d  4.16c,d  0.26  0.09  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. View Large Table 9. Effects of DHA-rich microalgae on antioxidant capacity in breast and thigh muscle of broilers. Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle:   Superoxide dismutase, U/mg  114b  162a  158a  10.9  0.05   Total antioxidant capacity, U/mg  5.63d  9.90c  9.01c,d  0.97  0.07   Glutathione peroxidase, U/mg  699  925  855  63.3  0.14   Catalase, U/mg  5.26b  9.78a  5.82b  0.80  0.03   Malondialdehyde, nmol/mg  5.64a  3.67b  4.35a,b  0.41  0.05  Thigh muscle:   Superoxide dismutase, U/mg  118b  185a  163a,b  16.1  0.05   Total antioxidant capacity, U/mg  5.71b  10.36a  7.61b  0.53  < 0.01   Glutathione peroxidase, U/mg  661b  853a,b  964a  55.2  0.04   Catalase, U/mg  6.36  9.25  6.15  0.91  0.13   Malondialdehyde, nmol/mg  4.67c  3.56d  4.16c,d  0.26  0.09  Item  CON1  1MA1  2MA1  SEM  P-value  Breast muscle:   Superoxide dismutase, U/mg  114b  162a  158a  10.9  0.05   Total antioxidant capacity, U/mg  5.63d  9.90c  9.01c,d  0.97  0.07   Glutathione peroxidase, U/mg  699  925  855  63.3  0.14   Catalase, U/mg  5.26b  9.78a  5.82b  0.80  0.03   Malondialdehyde, nmol/mg  5.64a  3.67b  4.35a,b  0.41  0.05  Thigh muscle:   Superoxide dismutase, U/mg  118b  185a  163a,b  16.1  0.05   Total antioxidant capacity, U/mg  5.71b  10.36a  7.61b  0.53  < 0.01   Glutathione peroxidase, U/mg  661b  853a,b  964a  55.2  0.04   Catalase, U/mg  6.36  9.25  6.15  0.91  0.13   Malondialdehyde, nmol/mg  4.67c  3.56d  4.16c,d  0.26  0.09  SEM, standard error of the mean. a,bDifferent superscripts within a row indicate a significant difference (P ≤ 0.05). c,dDifferent superscripts within a row indicate a tendency of difference (0.05< P ≤ 0.10). 1CON: 3% soybean oil, in the control group; 1MA: 1% microalgae + 2% soybean oil; 2MA: 2% microalgae + 1% soybean oil. View Large DISCUSSION In the present research, we demonstrated that birds fed diets supplemented with 1% and 2% MA showed improved BW, ADG, and lower FCR, which is in agreement with Kessi (2016). This result may be due to the positive effects of MA on improving serum composition and immune status (Abudabos et al., 2013), and decreasing microbial load in digestive tract (Zahid et al., 2001; Ali and Memon, 2008) of birds. However, birds fed with 2MA showed decreased ADFI even though ADG was increased, which might be related to the high content of fiber (polysaccharide) in the algal biomass (Armin et al., 2015), the high concentrations of phenolic compounds (De Lange, 2000), or the high levels of heavy metals, toxins, and arsenic in MA (Harinder et al., 2016). The improved thigh muscle weight and yield in our study is associated with the higher final BW of birds by MA (Abudabos et al., 2013; Ribeiro et al., 2013). However, unlike our study, Bharath et al. (2017) reported carcass traits like dressing yield, breast yield and liver percentage were not influenced by n-3 PUFA, which might be due to the different type and amount of MA used (Harinder et al., 2016). The decreased abdominal fat percentage by MA in our study is due to the positive effects of DHA in MA on utilization of lipid in serum (Gatrell et al., 2017), which is usually revealed by the decreased serum cholesterol and triglyceride levels (Armin et al., 2015), while our study also found the concentration of LDL-C in serum was reduced. The improved liver percentage and spleen percentage in our study were beneficial for the immunity in birds and n-3 PUFA deposition in chicken meat (Mariey et al., 2014), which might result in better growth performance. Moreover, these improved liver percentage and spleen percentage might be the reason for the increased serological index and higher content of n-3 PUFA in diets and muscle (Schreiner et al., 2005). Together with the improved carcass trait, birds also showed better serological index. Serum glucose is an important source of energy for animals and can be conducive to growth of body tissues, while serum concentrations of albumin and total protein reflect the functions of synthesis of proteins in liver of broilers, which may associate with animal growth and physiological status (Limdi and Hyde, 2003). In the present study, birds fed MA showed increased levels of serum glucose, total protein, albumin, and globulin, which was also revealed by Mariey et al. (2014). Furthermore, the MA-induced reduction in serum UN observed in our study suggests that MA helped birds achieve more efficient nitrogen utilization (Brown and Cline, 1974), while this nitrogen retention or nitrogen balance helps to the balance between body protein synthesis and body protein degradation in birds (Metayer et al., 2008). Except for the glucose and protein metabolism, the lipid utilization was also improved in our study, which were also revealed in the studies of Abudabos et al. (2013) and Mariey et al. (2014). In our study, birds fed with MA showed the lower fat content in serum of birds, especially TC, TG, and LDL-C (Wahbeh, 1997; Abudabos et al., 2013), which was associated with higher concentration of n-3 PUFA in diets and muscle, particularly EPA and DHA. Actually, DHA supplementation from MA reduced serum TG and increase HDL-C (Bernstein et al., 2011) because DHA can decrease TG levels by reducing hepatic very low-density lipoprotein (VLDL) synthesis (Roche and Gibney, 2000). Reduced synthesis in turn lead to reduced secretion and smaller VLDL particles, which were more readily converted to LDL and HDL (Mori et al., 2000). Cholesterol issues were usually addressed by lowering LDL-C, while increasing concentration of serum HDL-C was considered as an important therapeutic target for improvement of the lipid profile and some cardiovascular disease (Colla et al., 2008). Our results showed MA regulated blood lipid balance might be through promoting HDL-C synthesis and accelerating TC, TG, LDL-C metabolism. The improved serum composition in birds might help reduce the risk for cardiovascular disease in persons who consume the chicken meats from broilers fed diets containing MA. Along with the altered serological index, we found the antioxidant capacity and fatty acid concentration in breast and thigh meat were also increased, which is in line with Armin et al. (2015), who found that 1% MA elicited an enhancement in n-3 PUFA accumulation on thigh and breast meat of birds along with improved antioxidant status due to the positive effect of high level of DHA in MA. Our results showed MA increased the concentrations of C14:0, C16:0, and SFA in meat, which is possibly because MA contain higher concentrations of C14:0 (5.15%), C16:0 (60.1%), and SFA (67.9%) than SO, while C14:0, C16:0, and SFA are hard to be oxidant and easy to be deposited. The PUFA in MA is 29%, lower than SO (62%), but the n-3 PUFA in MA is 29%, higher than SO (8%), which indicates MA mainly contains n-3 PUFA, while SO mainly contains n-6 PUFA. In our study, we also found MA could lower the concentration of n-6 PUFA in diets, breast and thigh muscle of birds because MA contain nearly no n-6 PUFA. This chicken meat might be beneficial for humans who consume it because a higher intake of n-6 PUFA in meat might increase the risk of human gallstones (Sturdevant et al., 1973), reduce high-density lipoprotein concentrations (Mattson and Grundy, 1985), and inhibit immune system function (Rasmussen et al., 1994). The most important n-3 PUFA in human nutrition are α-Linolenic acid (C18:3n-3), EPA, and DHA (Burdge, 2004). Our study found that α-Linolenic acid were reduced along with the increased level of EPA and DHA, which is because α-Linolenic acid serves as a precursor for the synthesis of EPA and DHA in thigh and breast muscle of broilers. Considering this conversion is limited in humans (Garg et al., 2006), our study uses MA (containing 28.7% DHA) as a primary source of DHA to improve the concentration of DHA in chicken meat (Lopez-Ferrer et al., 2001a; Zelenka et al., 2008). Our study demonstrates that the fatty acid profiles in breast or thigh muscle are in line with these in diets, which is because broilers are normally in a positive energy balance and the PUFAs supplied through diets are deposited in tissues mostly unaltered, so the composition of carcass fat tends to reflect that of dietary fat (Lopez-Ferrer et al., 2001a; Betti et al., 2009; Ribeiro et al., 2013). The key to positive health effects of n-3 PUFA was a balanced ratio of n-6 to n-3 PUFA (Schreiner et al., 2005), which might reduce the risk of lifestyle diseases such as coronary artery disease. Burghardt et al. (2010) pointed out nutritional recommendations in human diets are that the P/S ratio should be above 0.45 and the n-6/n-3 PUFA ratio should not exceed 4, while Sugano (1996) found the recommended ratio of n-6/n-3 PUFA ratio in the human diets ranges between 3:1 and 6:1. Interestingly, these requirements are comparable to the ratios obtained in the breast and thigh meat of chickens fed MA in our study, so consumption of this chicken meat by humans might help reduce the risk of coronary artery atherosclerotic disease and some common adult cancers (Renehan et al., 2008; Jiang et al., 2013). Due to the nutritional and physiological characteristics, broilers are prone to lipid peroxidation, resulting in more peroxidative metabolites. Feeding birds with lipid-rich diet may cause oxidative stress, which may damage DNA, bio-membrane lipids, and proteins, as well as a variety of impairments to tissue of birds (Zhao and Shen, 2005; Zhang et al., 2011; Delles et al., 2014). The MDA is a major product of lipid peroxidation, and is an effective marker of oxidative stress (Lu et al., 2010). In our research, we found 1% or 2% MA was effective in decreasing level of MDA in meat mainly because of the DHA and antioxidants in MA. For one thing, the high concentration of DHA scavenges free radical and regulate the level of reactive oxygen species in vivo through the action of NAD (P) H oxidase or through its own peroxidation reaction and free radical reaction (Richard et al., 2008). The present study demonstrates DHA in MA reduce MDA via activing the enzyme and the non-enzyme antioxidant system. Enzymatic inactivation of reactive oxygen species in muscle tissue is mainly achieved by the higher concentration of SOD, CAT, and GSH-Px because SOD and CAT are antioxidant enzymes that directly react with radical species, whereas GSH-Px regenerates oxidized antioxidants (Delles et al., 2014). The non-enzymatic antioxidant defense system is reflected by the increased concentration of T-AOC (Wang et al., 2011). For another, MA had some antioxidants, including beta-carotene and vitamin E, which may play an important role in protecting endogenous lipids from peroxidation and oxidation. Our findings suggest that 1% or 2% MA (containing 2.04% vitamin A and 0.07% vitamin E) can increase antioxidant capacity of chicken meat. The vitamin A in our study may reflect the function of beta-carotene on producing high-oxidative stability chicken meat (Barclay et al., 1994) because beta-carotene is one of the provitamin A carotenoids (Olson, 1989). Furthermore, the MA in our study may have the same effect as vitamin E in lowering concentration of MDA and improving oxidative stability of broiler meat enriched with n-3 PUFA (Rymer et al., 2010; Armin et al., 2015). CONCLUSION These results demonstrate that supplementing diets with 1% or 2% microalgae (Schizochytrium limacinum CCAP 4087/2) replacing soybean oil effectively improved performance, carcass traits, serum composition, antioxidant capacity, and n-3 PUFA (EPA and DHA) deposition in breast and thigh muscle of broiler chickens. ACKNOWLEDGMENTS This research is supported by the National Natural Science Foundation of China (31772612) and CARS-35. We also acknowledge Alltech Inc. (Nicholasville, KY) for providing the novel microalgae products which enriches DHA. REFERENCES Abudabos A. M., Okab A. B., Aljumaah R. S., Samara E. M.. 2013. Nutritional value of green seaweed (ulva lactuca) for broiler chickens. Ital. J. Anim. Sci.  12: 612– 620. Adkins Y., Kelley D. S.. 2010. 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Poultry ScienceOxford University Press

Published: Mar 1, 2018

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