Evaluation of Phytase, Xylanase, and Protease in Reduced Nutrient Diets Fed to Broilers

Evaluation of Phytase, Xylanase, and Protease in Reduced Nutrient Diets Fed to Broilers Abstract Two experiments were conducted to evaluate phytase, phytase + xylanase, or phytase + xylanase + protease in diets deficient in Ca, available phosphorus (avP), energy, and amino acids. Lohman Indian River, straight-run broilers (n = 2,304 and 4,752 in experiments 1 and 2, respectively) were fed one of eight diets: (1) nutrient adequate control (PC), (2) negative control (NC) 1 with reduced Ca and avP, (3) NC1 + 1,500 FTU/kg phytase, (4) NC2 with reduced Ca, avP and energy, (5) NC2 + 1,500 FTU/kg phytase + 16,000 BXU/kg xylanase, (6) NC3 with reduced Ca, avP, energy, and amino acids, (7) NC3 + 1,500 FTU/kg phytase + 16,000 BXU/kg xylanase, (8) NC3 + 1,500 FTU/kg phytase + 16,000 BXU/kg xylanase + 15,000 U/kg protease. Reducing Ca, avP, energy, and amino acids significantly reduced body weight gain (BWG) and feed efficiency in a sequential fashion, with birds fed NC3 having a significantly higher FCR compared with birds fed NC1 or the PC. In both experiments, supplementation with phytase, phytase + xylanase, or the combination of phytase + xylanase + protease significantly improved BWG and FCR comparable to the PC. However, there was no difference in BWG or FCR between birds fed phytase + xylanase or phytase + xylanase + protease. Results indicate that supplementation of phytase + xylanase restored the BWG and FCR of the birds fed reduced Ca, avP, energy, and amino acid diets, and supplementation of protease to diets containing phytase and xylanase needs further investigation. DESCRIPTION OF PROBLEM The use of exogenous enzymes, specifically phytase and xylanase, is reported to save the global feed market an estimated 3–5 billion dollars per year [1]. Numerous research trials have evaluated the influence of enzymes individually or in combination on the growth performance and nutrient digestibility of broiler chicks. In most cases, the enzymes are applied to diets containing marginal levels of Ca, available phosphorus (avP), and energy, particularly when phytase, xylanase, amylase + protease, phytase + xylanase, or xylanase + protease are evaluated [2, 3, 4]. It is believed that each enzyme works independently to provide specific nutrients to the bird in an additive or sub-additive fashion. For example, protease is reported to increase amino acid digestibility, as well as have ancillary effects on starch, fat and digestible energy with inconsistent effects on growth performance as reviewed by Cowieson and Roos [5]. Supplementation of xylanase into corn-based diets is reported to increase fat and starch digestibility through improved digestion of resistant starch, improved access to cell walls via reduction in cell wall integrity, and/or modification of intestinal microbial communities through the production of prebiotic-like oligosaccharides [2]. Carbohydrases, such as xylanase, are also reported to increase apparent amino acid digestibility over the control diet by approximately 4.5%, which is reduced to 4.0% in the presence of phytase [6]. Phytase is known to hydrolyze phytate P in plants, thereby increasing the avP content of the diet and enabling reductions in added inorganic P, while at the same time reducing the anti-nutrient effects of phytate. This antinutritive effect extends to minerals, specifically Ca, allowing for reductions in Ca concentrations in the diet, and to energy and amino acids as reviewed by Dersjant-Li et al. [7]. However, the effect of phytase on energy and amino acid digestibility is less consistent than the effects of phytase on P. For example, Cowieson and Adeola [3] reported phytase had no effect on ileal digestible energy in broilers fed diets deficient in Ca, P, and energy (180 kcal/kg). In that same trial, performance tended to improve with phytase supplementation or supplementation of xylanase + amylase + protease, with the greatest performance achieved in the deficient diets when phytase was supplemented in combination with xylanase + amylase + protease. Questions remain regarding the benefits of supplementing broiler diets, marginal in Ca, avP, energy, and amino acids with phytase alone, or phytase + carbohydrases, or phytase + carbohydrase + protease to provide additional nutrients over and above that of a combination of phytase and carbohydrase. Therefore, the objective of this set of trials was to determine the effect of supplemental phytase alone and in combination with xylanase and protease on broiler performance when fed diets with reductions in Ca, avP, energy, and amino acids. MATERIALS AND METHODS The trial was conducted at Japfa Poultry Research Facilities under the guidelines of the ethical principles for use of animals for scientific purposes. Experiment 1 Straight-run broilers (n = 2,304) were obtained at hatch and housed in 128 battery cages in a closed house with tunnel ventilation [8]. There were 18 birds per cage and 16 replicate cages per treatment. Birds had ad libitum access to feed and water throughout the 21-d study. Starter diets were fed in crumble form. Birds and feed were weighed at the start (d 0) and end (d 21) of the experiment to determine feed intake (FI) and body weight gain (BWG) and calculate FCR. Culled or dead birds were recorded and weighed and FI was adjusted for bird days. Experiment 2 Straight-run broilers (n = 4,752) were obtained at hatch and housed in 72 floor pens [9]. There were 66 birds per pen and 9 replicate pens per treatment. Birds had ad libitum access to feed and water provided by bell feeders and drinkers for the 35-d study. Birds were fed crumble starter diets from hatch to 21 d and pelleted grower diets from 22 to 35 d post-hatch. Birds and feed were weighed at the start (d 0), d 21, and end (d 35) of the experiment to determine FI and BWG and calculate FCR. Culled or dead birds were recorded and weighed and FI was adjusted for bird days. Experimental Design and Statistics Four starter (experiments 1 and 2) and grower (experiment 2) basal diets (Tables 1 and 2) were mixed: (1) a nutrient adequate PC formulated to meet or exceed Rostagno et al. [10] nutrient requirements for 21 and 35-d-old broilers; (2) negative control (NC) 1, reduced in Ca by 0.20% and avP by 0.18% compared with the PC; (3) NC2, reduced in Ca by 0.20%, avP by 0.18%, and ME by 80 kcal/kg compared with the PC; and (4) NC3, reduced in Ca by 0.20%, avP by 0.18%, ME by 80 kcal/kg, and digestible amino acids by 5% compared with the PC. The remaining four experimental diets were as follows: (5) NC1 plus 1,500 FTU/kg phytase [11]; (6) NC2 plus 1,500 FTU/kg and 16,000 BXU/kg xylanase [12]; (7) NC3 plus 1,500 FTU/kg and 16,000 BXU/kg; and (8) NC3 plus 1,500 FTU/kg, 16,000 BXU/kg, and 15,000 U/kg protease [13]. Table 1. Ingredient and nutrient composition of the starter diets (as is basis). Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 55.28 54.46 53.81 55.43 Corn gluten meal 3.78 3.79 3.80 3.12 Corn dried distillers grains with solubles 4.50 4.50 4.50 4.50 Wheat bran 3.22 5.56 7.64 8.54 Soybean meal 25.94 25.45 25.01 23.16 Palm oil 2.80 2.80 1.80 1.80 Limestone 1.58 1.39 1.40 1.41 Monocalcium phosphate 1.36 0.50 0.49 0.50 Salt 0.23 0.23 0.23 0.23 L—lysine HCl 0.35 0.36 0.36 0.35 DL—methionine 0.25 0.25 0.25 0.23 L—threonine 0.09 0.09 0.10 0.09 Sodium bicarbonate 0.30 0.30 0.31 0.30 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.04 0.04 0.04 0.04 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.72 88.64 88.55 88.52 Fat 5.75 5.80 4.86 4.93 Fiber 2.65 2.84 3.02 3.05 Ash 5.99 5.20 5.26 5.21 Crude protein 21.33 21.38 21.43 20.40 ME, kcal/kg 3,000.00 3,000.00 2,920.00 2,920.00 Digestible lysine 1.15 1.15 1.15 1.09 Digestible methionine 0.57 0.57 0.56 0.53 Digestible TSAA 0.85 0.85 0.85 0.81 Digestible tryptophan 0.20 0.20 0.21 0.20 Digestible arginine 1.21 1.21 1.21 1.15 Digestible threonine 0.76 0.76 0.76 0.72 Digestible leucine 1.84 1.83 1.82 1.72 Digestible valine 0.88 0.88 0.87 0.83 Digestible isoleucine 0.78 0.78 0.78 0.73 Calcium 0.90 0.70 0.70 0.70 Total phosphorus 0.70 0.53 0.54 0.54 Available phosphorus 0.42 0.24 0.24 0.24 Sodium 0.19 0.19 0.19 0.19 Chloride 0.25 0.25 0.25 0.25 Choline 1,400.00 1,400.00 1,400.00 1,400.00 Analyzed nutrient composition, % Crude protein 21.4 21.4 21.5 20.07 Calcium 0.84 0.71 0.68 0.69 Total phosphorus 0.69 0.54 0.54 0.55 Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 55.28 54.46 53.81 55.43 Corn gluten meal 3.78 3.79 3.80 3.12 Corn dried distillers grains with solubles 4.50 4.50 4.50 4.50 Wheat bran 3.22 5.56 7.64 8.54 Soybean meal 25.94 25.45 25.01 23.16 Palm oil 2.80 2.80 1.80 1.80 Limestone 1.58 1.39 1.40 1.41 Monocalcium phosphate 1.36 0.50 0.49 0.50 Salt 0.23 0.23 0.23 0.23 L—lysine HCl 0.35 0.36 0.36 0.35 DL—methionine 0.25 0.25 0.25 0.23 L—threonine 0.09 0.09 0.10 0.09 Sodium bicarbonate 0.30 0.30 0.31 0.30 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.04 0.04 0.04 0.04 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.72 88.64 88.55 88.52 Fat 5.75 5.80 4.86 4.93 Fiber 2.65 2.84 3.02 3.05 Ash 5.99 5.20 5.26 5.21 Crude protein 21.33 21.38 21.43 20.40 ME, kcal/kg 3,000.00 3,000.00 2,920.00 2,920.00 Digestible lysine 1.15 1.15 1.15 1.09 Digestible methionine 0.57 0.57 0.56 0.53 Digestible TSAA 0.85 0.85 0.85 0.81 Digestible tryptophan 0.20 0.20 0.21 0.20 Digestible arginine 1.21 1.21 1.21 1.15 Digestible threonine 0.76 0.76 0.76 0.72 Digestible leucine 1.84 1.83 1.82 1.72 Digestible valine 0.88 0.88 0.87 0.83 Digestible isoleucine 0.78 0.78 0.78 0.73 Calcium 0.90 0.70 0.70 0.70 Total phosphorus 0.70 0.53 0.54 0.54 Available phosphorus 0.42 0.24 0.24 0.24 Sodium 0.19 0.19 0.19 0.19 Chloride 0.25 0.25 0.25 0.25 Choline 1,400.00 1,400.00 1,400.00 1,400.00 Analyzed nutrient composition, % Crude protein 21.4 21.4 21.5 20.07 Calcium 0.84 0.71 0.68 0.69 Total phosphorus 0.69 0.54 0.54 0.55 1Trace mineral premix provided per kg of diet: selenium 0.09 mg, iodine 0.18 mg, copper 3.00 mg, iron 36.0 mg, manganese 54.0 mg, zinc 48.0 mg, cobalt 0.12 mg. 2Vitamin premix provided per kg of diet: Vit A 11,550 IU, Vit D3 4,300 IU, Vit E 27.5 IU, Vit K3 3.85 mg, Vit B1 2.75 mg, Vit B2 9.9 mg, Vit B6 3.85 mg, Vit B12 22.0 Mcg, niacin 49.5 mg, pantothenic acid 15.4 mg, folic acid 1.38 mg, biotin 166 Mcg, selenium 0.08 mg, iodine 1.38 mg. View Large Table 1. Ingredient and nutrient composition of the starter diets (as is basis). Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 55.28 54.46 53.81 55.43 Corn gluten meal 3.78 3.79 3.80 3.12 Corn dried distillers grains with solubles 4.50 4.50 4.50 4.50 Wheat bran 3.22 5.56 7.64 8.54 Soybean meal 25.94 25.45 25.01 23.16 Palm oil 2.80 2.80 1.80 1.80 Limestone 1.58 1.39 1.40 1.41 Monocalcium phosphate 1.36 0.50 0.49 0.50 Salt 0.23 0.23 0.23 0.23 L—lysine HCl 0.35 0.36 0.36 0.35 DL—methionine 0.25 0.25 0.25 0.23 L—threonine 0.09 0.09 0.10 0.09 Sodium bicarbonate 0.30 0.30 0.31 0.30 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.04 0.04 0.04 0.04 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.72 88.64 88.55 88.52 Fat 5.75 5.80 4.86 4.93 Fiber 2.65 2.84 3.02 3.05 Ash 5.99 5.20 5.26 5.21 Crude protein 21.33 21.38 21.43 20.40 ME, kcal/kg 3,000.00 3,000.00 2,920.00 2,920.00 Digestible lysine 1.15 1.15 1.15 1.09 Digestible methionine 0.57 0.57 0.56 0.53 Digestible TSAA 0.85 0.85 0.85 0.81 Digestible tryptophan 0.20 0.20 0.21 0.20 Digestible arginine 1.21 1.21 1.21 1.15 Digestible threonine 0.76 0.76 0.76 0.72 Digestible leucine 1.84 1.83 1.82 1.72 Digestible valine 0.88 0.88 0.87 0.83 Digestible isoleucine 0.78 0.78 0.78 0.73 Calcium 0.90 0.70 0.70 0.70 Total phosphorus 0.70 0.53 0.54 0.54 Available phosphorus 0.42 0.24 0.24 0.24 Sodium 0.19 0.19 0.19 0.19 Chloride 0.25 0.25 0.25 0.25 Choline 1,400.00 1,400.00 1,400.00 1,400.00 Analyzed nutrient composition, % Crude protein 21.4 21.4 21.5 20.07 Calcium 0.84 0.71 0.68 0.69 Total phosphorus 0.69 0.54 0.54 0.55 Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 55.28 54.46 53.81 55.43 Corn gluten meal 3.78 3.79 3.80 3.12 Corn dried distillers grains with solubles 4.50 4.50 4.50 4.50 Wheat bran 3.22 5.56 7.64 8.54 Soybean meal 25.94 25.45 25.01 23.16 Palm oil 2.80 2.80 1.80 1.80 Limestone 1.58 1.39 1.40 1.41 Monocalcium phosphate 1.36 0.50 0.49 0.50 Salt 0.23 0.23 0.23 0.23 L—lysine HCl 0.35 0.36 0.36 0.35 DL—methionine 0.25 0.25 0.25 0.23 L—threonine 0.09 0.09 0.10 0.09 Sodium bicarbonate 0.30 0.30 0.31 0.30 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.04 0.04 0.04 0.04 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.72 88.64 88.55 88.52 Fat 5.75 5.80 4.86 4.93 Fiber 2.65 2.84 3.02 3.05 Ash 5.99 5.20 5.26 5.21 Crude protein 21.33 21.38 21.43 20.40 ME, kcal/kg 3,000.00 3,000.00 2,920.00 2,920.00 Digestible lysine 1.15 1.15 1.15 1.09 Digestible methionine 0.57 0.57 0.56 0.53 Digestible TSAA 0.85 0.85 0.85 0.81 Digestible tryptophan 0.20 0.20 0.21 0.20 Digestible arginine 1.21 1.21 1.21 1.15 Digestible threonine 0.76 0.76 0.76 0.72 Digestible leucine 1.84 1.83 1.82 1.72 Digestible valine 0.88 0.88 0.87 0.83 Digestible isoleucine 0.78 0.78 0.78 0.73 Calcium 0.90 0.70 0.70 0.70 Total phosphorus 0.70 0.53 0.54 0.54 Available phosphorus 0.42 0.24 0.24 0.24 Sodium 0.19 0.19 0.19 0.19 Chloride 0.25 0.25 0.25 0.25 Choline 1,400.00 1,400.00 1,400.00 1,400.00 Analyzed nutrient composition, % Crude protein 21.4 21.4 21.5 20.07 Calcium 0.84 0.71 0.68 0.69 Total phosphorus 0.69 0.54 0.54 0.55 1Trace mineral premix provided per kg of diet: selenium 0.09 mg, iodine 0.18 mg, copper 3.00 mg, iron 36.0 mg, manganese 54.0 mg, zinc 48.0 mg, cobalt 0.12 mg. 2Vitamin premix provided per kg of diet: Vit A 11,550 IU, Vit D3 4,300 IU, Vit E 27.5 IU, Vit K3 3.85 mg, Vit B1 2.75 mg, Vit B2 9.9 mg, Vit B6 3.85 mg, Vit B12 22.0 Mcg, niacin 49.5 mg, pantothenic acid 15.4 mg, folic acid 1.38 mg, biotin 166 Mcg, selenium 0.08 mg, iodine 1.38 mg. View Large Table 2. Ingredient and nutrient composition of the grower diets (as is basis). Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 62.05 61.23 60.59 62.05 Corn gluten meal 3.60 3.61 3.61 2.93 Corn dried distillers grains with solubles 6.00 6.00 6.00 6.00 Wheat bran 3.02 5.35 7.43 8.12 Soybean meal 18.60 18.11 17.67 16.20 Palm oil 3.00 3.00 2.00 2.00 Limestone 1.38 1.19 1.20 1.20 Monocalcium phosphate 1.02 0.16 0.15 0.16 Salt 0.18 0.18 0.18 0.18 L—lysine HCl 0.32 0.33 0.33 0.32 DL—methionine 0.17 0.17 0.17 0.16 L—threonine 0.07 0.07 0.07 0.07 Sodium bicarbonate 0.28 0.29 0.29 0.29 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.05 0.05 0.04 0.05 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.62 88.53 88.45 88.41 Fat 6.22 6.28 5.34 5.40 Fiber 2.56 2.75 2.92 2.95 Ash 5.12 4.33 4.40 4.35 Crude protein 18.54 18.59 18.64 17.75 ME, kcal/kg 3,100.00 3,100.00 3,020.00 3,020.00 Digestible lysine 0.95 0.95 0.95 0.90 Digestible methionine 0.46 0.46 0.46 0.44 Digestible TSAA 0.72 0.72 0.72 0.69 Digestible tryptophan 0.17 0.17 0.17 0.16 Digestible arginine 1.00 1.00 1.00 0.95 Digestible threonine 0.64 0.64 0.64 0.61 Digestible valine 0.76 0.76 0.76 0.72 Digestible leucine 1.67 1.66 1.66 1.56 Digestible isoleucine 0.66 0.66 0.65 0.61 Calcium 0.75 0.55 0.55 0.55 Total phosphorus 0.60 0.43 0.44 0.44 Available phosphorus 0.34 0.16 0.16 0.16 Sodium 0.17 0.17 0.17 0.17 Chloride 0.22 0.22 0.22 0.22 Choline 1,300.00 1,300.00 1,300.00 1,300.00 Analyzed nutrient composition, % Crude protein 18.3 18.9 18.6 17.7 Calcium 0.67 0.54 0.54 0.56 Total phosphorus 0.58 0.44 0.45 0.44 Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 62.05 61.23 60.59 62.05 Corn gluten meal 3.60 3.61 3.61 2.93 Corn dried distillers grains with solubles 6.00 6.00 6.00 6.00 Wheat bran 3.02 5.35 7.43 8.12 Soybean meal 18.60 18.11 17.67 16.20 Palm oil 3.00 3.00 2.00 2.00 Limestone 1.38 1.19 1.20 1.20 Monocalcium phosphate 1.02 0.16 0.15 0.16 Salt 0.18 0.18 0.18 0.18 L—lysine HCl 0.32 0.33 0.33 0.32 DL—methionine 0.17 0.17 0.17 0.16 L—threonine 0.07 0.07 0.07 0.07 Sodium bicarbonate 0.28 0.29 0.29 0.29 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.05 0.05 0.04 0.05 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.62 88.53 88.45 88.41 Fat 6.22 6.28 5.34 5.40 Fiber 2.56 2.75 2.92 2.95 Ash 5.12 4.33 4.40 4.35 Crude protein 18.54 18.59 18.64 17.75 ME, kcal/kg 3,100.00 3,100.00 3,020.00 3,020.00 Digestible lysine 0.95 0.95 0.95 0.90 Digestible methionine 0.46 0.46 0.46 0.44 Digestible TSAA 0.72 0.72 0.72 0.69 Digestible tryptophan 0.17 0.17 0.17 0.16 Digestible arginine 1.00 1.00 1.00 0.95 Digestible threonine 0.64 0.64 0.64 0.61 Digestible valine 0.76 0.76 0.76 0.72 Digestible leucine 1.67 1.66 1.66 1.56 Digestible isoleucine 0.66 0.66 0.65 0.61 Calcium 0.75 0.55 0.55 0.55 Total phosphorus 0.60 0.43 0.44 0.44 Available phosphorus 0.34 0.16 0.16 0.16 Sodium 0.17 0.17 0.17 0.17 Chloride 0.22 0.22 0.22 0.22 Choline 1,300.00 1,300.00 1,300.00 1,300.00 Analyzed nutrient composition, % Crude protein 18.3 18.9 18.6 17.7 Calcium 0.67 0.54 0.54 0.56 Total phosphorus 0.58 0.44 0.45 0.44 1Trace mineral premix provided per kg of diet: selenium 0.09 mg, iodine 0.18 mg, copper 3.00 mg, iron 36.0 mg, manganese 54.0 mg, zinc 48.0 mg, cobalt 0.12 mg. 2Vitamin premix provided per kg of diet: Vit A 9,450 IU, Vit D3 3,700 IU, Vit E 22.5 IU, Vit K3 3.15 mg, Vit B1 2.25 mg, Vit B2 8.1 mg, Vit B6 3.15 mg, Vit B12 18.0 Mcg, niacin 40.5 mg, pantothenic acid 12.6 mg, folic acid 1.13 mg, biotin 116 Mcg, selenium 0.07 mg, iodine 1.13 mg. View Large Table 2. Ingredient and nutrient composition of the grower diets (as is basis). Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 62.05 61.23 60.59 62.05 Corn gluten meal 3.60 3.61 3.61 2.93 Corn dried distillers grains with solubles 6.00 6.00 6.00 6.00 Wheat bran 3.02 5.35 7.43 8.12 Soybean meal 18.60 18.11 17.67 16.20 Palm oil 3.00 3.00 2.00 2.00 Limestone 1.38 1.19 1.20 1.20 Monocalcium phosphate 1.02 0.16 0.15 0.16 Salt 0.18 0.18 0.18 0.18 L—lysine HCl 0.32 0.33 0.33 0.32 DL—methionine 0.17 0.17 0.17 0.16 L—threonine 0.07 0.07 0.07 0.07 Sodium bicarbonate 0.28 0.29 0.29 0.29 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.05 0.05 0.04 0.05 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.62 88.53 88.45 88.41 Fat 6.22 6.28 5.34 5.40 Fiber 2.56 2.75 2.92 2.95 Ash 5.12 4.33 4.40 4.35 Crude protein 18.54 18.59 18.64 17.75 ME, kcal/kg 3,100.00 3,100.00 3,020.00 3,020.00 Digestible lysine 0.95 0.95 0.95 0.90 Digestible methionine 0.46 0.46 0.46 0.44 Digestible TSAA 0.72 0.72 0.72 0.69 Digestible tryptophan 0.17 0.17 0.17 0.16 Digestible arginine 1.00 1.00 1.00 0.95 Digestible threonine 0.64 0.64 0.64 0.61 Digestible valine 0.76 0.76 0.76 0.72 Digestible leucine 1.67 1.66 1.66 1.56 Digestible isoleucine 0.66 0.66 0.65 0.61 Calcium 0.75 0.55 0.55 0.55 Total phosphorus 0.60 0.43 0.44 0.44 Available phosphorus 0.34 0.16 0.16 0.16 Sodium 0.17 0.17 0.17 0.17 Chloride 0.22 0.22 0.22 0.22 Choline 1,300.00 1,300.00 1,300.00 1,300.00 Analyzed nutrient composition, % Crude protein 18.3 18.9 18.6 17.7 Calcium 0.67 0.54 0.54 0.56 Total phosphorus 0.58 0.44 0.45 0.44 Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 62.05 61.23 60.59 62.05 Corn gluten meal 3.60 3.61 3.61 2.93 Corn dried distillers grains with solubles 6.00 6.00 6.00 6.00 Wheat bran 3.02 5.35 7.43 8.12 Soybean meal 18.60 18.11 17.67 16.20 Palm oil 3.00 3.00 2.00 2.00 Limestone 1.38 1.19 1.20 1.20 Monocalcium phosphate 1.02 0.16 0.15 0.16 Salt 0.18 0.18 0.18 0.18 L—lysine HCl 0.32 0.33 0.33 0.32 DL—methionine 0.17 0.17 0.17 0.16 L—threonine 0.07 0.07 0.07 0.07 Sodium bicarbonate 0.28 0.29 0.29 0.29 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.05 0.05 0.04 0.05 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.62 88.53 88.45 88.41 Fat 6.22 6.28 5.34 5.40 Fiber 2.56 2.75 2.92 2.95 Ash 5.12 4.33 4.40 4.35 Crude protein 18.54 18.59 18.64 17.75 ME, kcal/kg 3,100.00 3,100.00 3,020.00 3,020.00 Digestible lysine 0.95 0.95 0.95 0.90 Digestible methionine 0.46 0.46 0.46 0.44 Digestible TSAA 0.72 0.72 0.72 0.69 Digestible tryptophan 0.17 0.17 0.17 0.16 Digestible arginine 1.00 1.00 1.00 0.95 Digestible threonine 0.64 0.64 0.64 0.61 Digestible valine 0.76 0.76 0.76 0.72 Digestible leucine 1.67 1.66 1.66 1.56 Digestible isoleucine 0.66 0.66 0.65 0.61 Calcium 0.75 0.55 0.55 0.55 Total phosphorus 0.60 0.43 0.44 0.44 Available phosphorus 0.34 0.16 0.16 0.16 Sodium 0.17 0.17 0.17 0.17 Chloride 0.22 0.22 0.22 0.22 Choline 1,300.00 1,300.00 1,300.00 1,300.00 Analyzed nutrient composition, % Crude protein 18.3 18.9 18.6 17.7 Calcium 0.67 0.54 0.54 0.56 Total phosphorus 0.58 0.44 0.45 0.44 1Trace mineral premix provided per kg of diet: selenium 0.09 mg, iodine 0.18 mg, copper 3.00 mg, iron 36.0 mg, manganese 54.0 mg, zinc 48.0 mg, cobalt 0.12 mg. 2Vitamin premix provided per kg of diet: Vit A 9,450 IU, Vit D3 3,700 IU, Vit E 22.5 IU, Vit K3 3.15 mg, Vit B1 2.25 mg, Vit B2 8.1 mg, Vit B6 3.15 mg, Vit B12 18.0 Mcg, niacin 40.5 mg, pantothenic acid 12.6 mg, folic acid 1.13 mg, biotin 116 Mcg, selenium 0.07 mg, iodine 1.13 mg. View Large Phytase recoveries in the diets were analyzed using a modified method of Engelen et al. [14]. Xylanase recovery in the experimental diets were analyzed using in house methods [15] and protease activity was analyzed using casein as a substrate [16]. Experimental diets were also analyzed for Ca [17] and total P [18], and crude protein [19]. Finally, data were arranged as a completely randomized design and analyzed as a 1-way ANOVA using the fit model platform in JMP Pro [20]. The model included diet. Pen served as the experimental unit. Data are presented as least square means per treatment group. Significant differences between the means were separated using multiple t-tests and significance was accepted at P < 0.05. RESULTS AND DISCUSSION Experimental Diets Nutrient analyses in the diets were similar to formulated (Table 1). However, phytase recoveries in the diets containing phytase (expected 1,500 FTU/kg) were approximately 50% lower than expected at 965, 698, 543, and 706 FTU/kg in the starter diets and 980, 862, 1,150, and 867 FTU/kg in the grower diets. The phytase activity of the premix analyzed as expected, at 6,590 FTU/g, when overages in the product from the manufacturer are considered. Xylanase recovery in the diets containing xylanase was approximately 17% higher than formulated and this is within the expected ranges from the manufacturer. Protease recovery in the diets was not performed due to lack of a suitable in feed assay. However, protease activity in the enzyme sample was analyzed at 4,42,000 U/g as described previously [16]. The expected activity of the protease was 75,000 protease U/g based on manufacturer assay recommendations [21], which were different from the assay used to determine protease activity in this experiment and may explain the differences reported between this paper and the manufacturer expectations. Experiment 1 Overall mortality was low (2.43%) and not associated (P = 0.81) with experimental diets (Table 3). Feed intake and BWG were reduced (P < 0.0001) in birds fed the NC diets compared with birds fed all other diets (Table 3). This has been previously reported in corn-soy diets fed to broilers marginally deficient in energy and essential amino acids [2], and in corn-soy diets containing wheat or rye and deficient in Ca, non-phytate P, and ME [3,4]. Supplementation of the NC2 or NC3 with phytase + xylanase or phytase + xylanase + protease increased (P < 0.0001) FI and BWG above that of birds fed the NCs and comparable to birds fed the PC (Table 3). Body weight gain was significantly higher in birds fed NC1 + 1,500 FTU/kg phytase compared with birds fed the PC (Table 3). In addition, supplementation of NC1 with 1,500 FTU/kg of phytase improved FCR compared with all other diets, except birds fed NC2 with phytase + xylanase, and this was comparable to the PC (Table 3). Feed conversion ratio was highest (P < 0.0001) in birds fed NC3 without any enzyme supplementation compared to all other diets, confirming the reduction in Ca, avP, energy, and amino acids significantly impacted FCR (Table 3). Supplementation of NC3 with phytase + xylanase significantly improved FCR and this was comparable to the PC, NC1, or NC2. Addition of protease to the phytase and xylanase combination had no additional benefit on FCR. Yuan et al. [22] reported significant improvements in FCR of 21-d-old broilers fed nutrient adequate corn-soy diets supplemented with carbohydrases (xylanase, beta-glucanase, and cellulase) with no further improvements in FCR with the addition of various doses of an added protease. Others have reported significantly improved BWG and FCR of 21-d-old broilers fed xylanase, protease, and amylase supplemented corn-soy diets whether they were formulated to be nutrient adequate or marginally deficient in energy and essential amino acids [2]. However, the enzymes in the previous trial were not evaluated individually or in combination with a phytase, and others have reported 21-d-old broilers fed corn, wheat, soy diets deficient in Ca, non-phytate P and ME gained more and were more efficient when the diets were supplemented with 1,000 FTU/kg of phytase regardless of the inclusion of an enzyme cocktail containing xylanase, amylase, or protease [4]. Table 3. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 21 d post-hatch (experiment 1) Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,301.3a 992.7b 1.299b,c 1.70 Negative control 12 0 0 0 1,185.8b 898.5c 1.311b 2.80 1,500 0 0 1,311.7a 1017.6a 1.269d 2.10 Negative control 23 0 0 0 1,178.2b 887.3c 1.314b 1.70 1,500 16,000 0 1,302.9a 1,000.3a,b 1.284c,d 3.10 Negative control 34 0 0 0 1,203.2b 892.2c 1.337a 3.50 1,500 16,000 0 1,311.4a 1,009.2a,b 1.304b 1.40 1,500 16,000 15,000 1,330.2a 1,010.5a,b 1.304b 3.10 SEM 12.0 8.2 0.01 1.1 P-value <0.0001 <0.0001 <0.0001 0.81 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,301.3a 992.7b 1.299b,c 1.70 Negative control 12 0 0 0 1,185.8b 898.5c 1.311b 2.80 1,500 0 0 1,311.7a 1017.6a 1.269d 2.10 Negative control 23 0 0 0 1,178.2b 887.3c 1.314b 1.70 1,500 16,000 0 1,302.9a 1,000.3a,b 1.284c,d 3.10 Negative control 34 0 0 0 1,203.2b 892.2c 1.337a 3.50 1,500 16,000 0 1,311.4a 1,009.2a,b 1.304b 1.40 1,500 16,000 15,000 1,330.2a 1,010.5a,b 1.304b 3.10 SEM 12.0 8.2 0.01 1.1 P-value <0.0001 <0.0001 <0.0001 0.81 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large Table 3. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 21 d post-hatch (experiment 1) Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,301.3a 992.7b 1.299b,c 1.70 Negative control 12 0 0 0 1,185.8b 898.5c 1.311b 2.80 1,500 0 0 1,311.7a 1017.6a 1.269d 2.10 Negative control 23 0 0 0 1,178.2b 887.3c 1.314b 1.70 1,500 16,000 0 1,302.9a 1,000.3a,b 1.284c,d 3.10 Negative control 34 0 0 0 1,203.2b 892.2c 1.337a 3.50 1,500 16,000 0 1,311.4a 1,009.2a,b 1.304b 1.40 1,500 16,000 15,000 1,330.2a 1,010.5a,b 1.304b 3.10 SEM 12.0 8.2 0.01 1.1 P-value <0.0001 <0.0001 <0.0001 0.81 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,301.3a 992.7b 1.299b,c 1.70 Negative control 12 0 0 0 1,185.8b 898.5c 1.311b 2.80 1,500 0 0 1,311.7a 1017.6a 1.269d 2.10 Negative control 23 0 0 0 1,178.2b 887.3c 1.314b 1.70 1,500 16,000 0 1,302.9a 1,000.3a,b 1.284c,d 3.10 Negative control 34 0 0 0 1,203.2b 892.2c 1.337a 3.50 1,500 16,000 0 1,311.4a 1,009.2a,b 1.304b 1.40 1,500 16,000 15,000 1,330.2a 1,010.5a,b 1.304b 3.10 SEM 12.0 8.2 0.01 1.1 P-value <0.0001 <0.0001 <0.0001 0.81 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large Experiment 2 Overall mortality (d 0–35) was 4.22% and not associated (P = 0.17) with diet (Tables 4 and 5). From d 0 to 21 (Table 4) and overall (Table 5), FI was reduced (P < 0.0001) in birds fed the NC diets compared with birds fed all other diets. Body weight gain from d 0 to 21 (Table 4) was lowest in birds fed NC3 compared with birds fed all other diets, except birds fed NC2 and this was comparable to birds fed NC1. Overall (d 0–35), BWG was reduced (P < 0.0001) in birds fed the NC diets compared to birds fed all other diets and enzyme supplementation in the NC diets improved BWG comparable to the PC (Table 5). As reported in experiment 1, FCR from d 0 to 21 was highest (P < 0.0001) in birds fed NC3 without enzyme supplementation compared with birds fed all other diets, except birds fed NC2 and this was similar to birds fed NC1 (Table 4). This suggests that the additional amino acid reduction in the NC diet had a real effect on performance over and above that of Ca, avP, and energy. Birds fed the PC had the best FCR and phytase supplementation in NC1 or phytase + xylanase supplementation in NC2 improved FCR comparable to birds fed the PC. Phytase + xylanase or phytase + xylanase + protease in NC3 improved FCR compared to birds fed NC3 without enzymes, but not comparable to the PC at d 21. However, overall (d 0–35) phytase + xylanase or the combination of phytase + xylanase + protease supplemented in NC3 improved (P < 0.0001) FCR compared with birds fed NC3 without enzymes and comparable to birds fed the PC, NC1 + phytase, or NC2 + phytase + xylanase (Table 5). Birds fed NC3 without enzymes had the highest FCR and this was not different than birds fed NC2 without enzymes, which was not different than birds fed NC1 without enzymes. As mentioned earlier, previous authors have reported significant reductions in performance when birds were fed diets deficient in Ca, avP, energy, and essential amino acids and xylanase + protease + amylase [2] or phytase alone [4] were able to improve growth performance in these low nutrient density diets. Table 4. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 21 d post-hatch (experiment 2) Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,386.8a 1,035.0a,b 1.331d 2.20 Negative control 12 0 0 0 1,232.0b 902.0c 1.361b,c 1.50 1,500 0 0 1,414.5a 1,050.5a 1.339c,d 1.50 Negative control 23 0 0 0 1,209.2b 870.5c,d 1.382a,b 2.40 1,500 16,000 0 1,404.4a 1,033.9a,b 1.357b,c,d 0.50 Negative control 34 0 0 0 1,216.3b 865.1d 1.402a 1.50 1,500 16,000 0 1,385.7a 1,014.5b 1.363b,c 1.20 1,500 16,000 15,000 1,411.2a 1,035.4a,b 1.362b,c 0.70 SEM 14.5 12.0 0.01 0.5 P-value <0.0001 <0.0001 <0.0001 0.11 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,386.8a 1,035.0a,b 1.331d 2.20 Negative control 12 0 0 0 1,232.0b 902.0c 1.361b,c 1.50 1,500 0 0 1,414.5a 1,050.5a 1.339c,d 1.50 Negative control 23 0 0 0 1,209.2b 870.5c,d 1.382a,b 2.40 1,500 16,000 0 1,404.4a 1,033.9a,b 1.357b,c,d 0.50 Negative control 34 0 0 0 1,216.3b 865.1d 1.402a 1.50 1,500 16,000 0 1,385.7a 1,014.5b 1.363b,c 1.20 1,500 16,000 15,000 1,411.2a 1,035.4a,b 1.362b,c 0.70 SEM 14.5 12.0 0.01 0.5 P-value <0.0001 <0.0001 <0.0001 0.11 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20% and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large Table 4. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 21 d post-hatch (experiment 2) Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,386.8a 1,035.0a,b 1.331d 2.20 Negative control 12 0 0 0 1,232.0b 902.0c 1.361b,c 1.50 1,500 0 0 1,414.5a 1,050.5a 1.339c,d 1.50 Negative control 23 0 0 0 1,209.2b 870.5c,d 1.382a,b 2.40 1,500 16,000 0 1,404.4a 1,033.9a,b 1.357b,c,d 0.50 Negative control 34 0 0 0 1,216.3b 865.1d 1.402a 1.50 1,500 16,000 0 1,385.7a 1,014.5b 1.363b,c 1.20 1,500 16,000 15,000 1,411.2a 1,035.4a,b 1.362b,c 0.70 SEM 14.5 12.0 0.01 0.5 P-value <0.0001 <0.0001 <0.0001 0.11 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,386.8a 1,035.0a,b 1.331d 2.20 Negative control 12 0 0 0 1,232.0b 902.0c 1.361b,c 1.50 1,500 0 0 1,414.5a 1,050.5a 1.339c,d 1.50 Negative control 23 0 0 0 1,209.2b 870.5c,d 1.382a,b 2.40 1,500 16,000 0 1,404.4a 1,033.9a,b 1.357b,c,d 0.50 Negative control 34 0 0 0 1,216.3b 865.1d 1.402a 1.50 1,500 16,000 0 1,385.7a 1,014.5b 1.363b,c 1.20 1,500 16,000 15,000 1,411.2a 1,035.4a,b 1.362b,c 0.70 SEM 14.5 12.0 0.01 0.5 P-value <0.0001 <0.0001 <0.0001 0.11 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20% and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large Table 5. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 35 d post-hatch (experiment 2). Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 3,710.1a 2,272.4a 1.612d 4.20 Negative control 12 0 0 0 3,498.4b 2,075.8b 1.664b,c 3.70 1,500 0 0 3,810.9a 2,332.0a 1.600d 4.90 Negative control 23 0 0 0 3,447.0b 1,992.1b 1.700a,b 6.20 1,500 16,000 0 3,810.6a 2,306.5a 1.612d 4.00 Negative control 34 0 0 0 3,464.5b 1,991.8b 1.722a 3.90 1,500 16,000 0 3,728.5a 2,263.2a 1.615d 4.20 1,500 16,000 15,000 3,742.4a 2,274.9a 1.625c,d 2.70 SEM 50.6 36.6 0.02 P-value <0.0001 <0.0001 <0.0001 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 3,710.1a 2,272.4a 1.612d 4.20 Negative control 12 0 0 0 3,498.4b 2,075.8b 1.664b,c 3.70 1,500 0 0 3,810.9a 2,332.0a 1.600d 4.90 Negative control 23 0 0 0 3,447.0b 1,992.1b 1.700a,b 6.20 1,500 16,000 0 3,810.6a 2,306.5a 1.612d 4.00 Negative control 34 0 0 0 3,464.5b 1,991.8b 1.722a 3.90 1,500 16,000 0 3,728.5a 2,263.2a 1.615d 4.20 1,500 16,000 15,000 3,742.4a 2,274.9a 1.625c,d 2.70 SEM 50.6 36.6 0.02 P-value <0.0001 <0.0001 <0.0001 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large Table 5. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 35 d post-hatch (experiment 2). Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 3,710.1a 2,272.4a 1.612d 4.20 Negative control 12 0 0 0 3,498.4b 2,075.8b 1.664b,c 3.70 1,500 0 0 3,810.9a 2,332.0a 1.600d 4.90 Negative control 23 0 0 0 3,447.0b 1,992.1b 1.700a,b 6.20 1,500 16,000 0 3,810.6a 2,306.5a 1.612d 4.00 Negative control 34 0 0 0 3,464.5b 1,991.8b 1.722a 3.90 1,500 16,000 0 3,728.5a 2,263.2a 1.615d 4.20 1,500 16,000 15,000 3,742.4a 2,274.9a 1.625c,d 2.70 SEM 50.6 36.6 0.02 P-value <0.0001 <0.0001 <0.0001 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 3,710.1a 2,272.4a 1.612d 4.20 Negative control 12 0 0 0 3,498.4b 2,075.8b 1.664b,c 3.70 1,500 0 0 3,810.9a 2,332.0a 1.600d 4.90 Negative control 23 0 0 0 3,447.0b 1,992.1b 1.700a,b 6.20 1,500 16,000 0 3,810.6a 2,306.5a 1.612d 4.00 Negative control 34 0 0 0 3,464.5b 1,991.8b 1.722a 3.90 1,500 16,000 0 3,728.5a 2,263.2a 1.615d 4.20 1,500 16,000 15,000 3,742.4a 2,274.9a 1.625c,d 2.70 SEM 50.6 36.6 0.02 P-value <0.0001 <0.0001 <0.0001 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large The BWG or FCR results from two separate trials confirm reducing Ca and avP as well as energy and amino acids was sufficient to reduce BWG and increase FCR in broilers, especially during the starter phase and this continued to market age. Enzyme supplementation in the NCs significantly improved BWG comparable to the PC and this was regardless of the enzymes, the combination, or the NC basal diet. Previous authors have reported carbohydrase (xylanase, beta-glucanase, and cellulase) supplementation in a nutrient adequate diet, with or without the addition of various doses of an acid protease, had no effect on gain of 21-d-old broilers, but resulted in significant improvements in FCR [22]. Reducing energy by 80 kcal in the low Ca and avP diets resulted in further reductions in FCR and supplementation of this diet with phytase + xylanase was able to improve FCR comparable to the PC in both experiments. Previous authors have reported significant improvements in broiler growth performance and/or feed efficiency with phytase [4], xylanase [22], or phytase + xylanase [23] with limited or no improvements in performance reported with the addition of other enzymes. Phytase and xylanase are reported to have sub-additive or additive benefits of in corn soy-based diets [6, 24] as the enzymes may act independently on different substrates. However, their effect on nutrients such as amino acids overlaps and are largely dependent on the digestible fraction or the nutritional value of the control diet to which the enzymes are added [6]. This may also contribute to the lack of an effect with the addition of proteases in the current trial, as the effect of protease is also reported to be dependent on the amino acid digestibility of the control diet [25], which is influenced by phytase dose [6, 26] and with the use of a carbohydrase [6]. Research and interest in protease supplementation in poultry diets has increased substantially in the past 5 years, with benefits in performance and/or amino acid digestibility being reported [5]. However, when considering the benefits of any enzyme supplementation, the response is largely dependent on the digestibility of the control diet. Given the majority of commercial poultry diets contain either a phytase or a carbohydrase and most often a combination of the two; it would be justified to further evaluate the supplementation of proteases in the presence of enzymes commonly used in the field and in a true factorial approach [27]. Recently, Santos et al. [28] tested standard or high doses of phytase, plus or minus xylanase, plus or minus protease in a true factorial design and reported significant improvements in FCR of 21-d-old broilers fed protease, but this effect was lost by day 42 and there was no significant benefit of the enzyme combinations on performance or carcass parameters. Similarly, it is clear from the current study that when the protease was used in a diet containing high doses of phytase and a xylanase, its benefit was not noticed. A recent meta-analysis on protease efficacy in broiler diets indicated only 19% of the protease trials published were conducted in the presence of a phytase and/or carbohydrase [29]. Further work into the benefits of protease in diets containing phytase, particularly at high doses, and a carbohydrase is warranted. CONCLUSIONS AND APPLICATIONS Birds fed diets at reduced levels of Ca, avP, ME, and amino acids had the worst performance, particularly FCR, indicating the reduction of these nutrients was inhibiting optimum growth. Supplementation of the low Ca, avP, ME, and amino acid diet with phytase + xylanase or phytase + xylanase + protease was able to improve BWG and FCR comparable to a nutrient adequate PC diet. There were no differences between the diets supplemented with phytase + xylanase or the diets supplemented with phytase + xylanase + protease. This lack of an effect of protease in a diet containing a phytase and xylanase is assumed to be related to the inherent digestibility of the control diet, which would be high in the presence of a phytase and xylanase. The benefits of supplementing protease enzymes in diets containing phytase and xylanase requires further evaluation and justification in corn soy-based diets with inherently high digestibility. Footnotes Primary Audience: Nutritionists, Formulators, Production Personnel, Scientists REFERENCES 1. Adeola O. , Cowieson A. J. . 2011 . Opportunities and challenges in using exogenous enzymes to improve nonruminant animal production . J. Anim. Sci. 89 : 3189 – 3218 . Google Scholar CrossRef Search ADS PubMed 2. Cowieson A. J. , Ravindran V. . 2008 . Effect of exogenous enzymes in maize-based diets varying in nutrient density for young broilers: growth performance and digestibility of energy, minerals and amino acids . Br. Poult. Sci. 49 : 37 – 44 . Google Scholar CrossRef Search ADS PubMed 3. Cowieson A. J. , Adeloa O. . 2005 . Carbohydrases, protease, and phytase have an additive beneficial effect in nutritionally marginal diets for broiler chicks . Poult. Sci. 84 : 1860 – 1867 . Google Scholar CrossRef Search ADS PubMed 4. Olukosi O. A. , Cowieson A. J. , Adeloa O. . 2008 . Energy utilization and growth performance of broilers receiving diets supplemented with enzymes containing carbohydrase or phytase activity individually or in combination . Br. J. Nutr. 99 : 682 – 690 . Google Scholar CrossRef Search ADS PubMed 5. Cowieson A. J. , Roos F. F. . 2016 . Toward optimal value creation through the application of exogenous mono-component protease in the diets of non-ruminants . Anim. Feed Sci. Tech. 221 : 331 – 340 . Google Scholar CrossRef Search ADS 6. Cowieson A. J. , Bedford M. R. . 2009 . The effect of phytase and carbohydrase on ileal amino acid digestibility in monogastric diets: complimentary mode of action? Worlds Poult. Sci. J. 65 : 609 – 624 . Google Scholar CrossRef Search ADS 7. Dersjant-Li Y. , Awati A. , Schulze H. , Partridge G. . 2015 . Phytase in non-ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors . J. Sci. Food Agric. 95 : 878 – 896 . Google Scholar CrossRef Search ADS PubMed 8. Indian River broilers (Aviagen, Indonesia) were housed in cages of galvanized steel with trough feeders, nipple drinkers, and raised wire floors. The lighting program was designed to provide 23:1 L:D (0-7 days) and 20:4:D (8-35 days). 9. Indian River broilers (Aviagen, Indonesia) were housed in floor pens with new rice hull litter in a closed house with tunnel ventilation The lighting program was designed to provide 23:1 L:D (0-7 days) and 20:4 L:D (8-35 days) . 10. Rostagno H. S. , Teixeira A. , Donzele J. L. , Gomes P. C. , De Oliveira R. F. M. , Lopes D. C. , Ferreira A. J. P. , Toledo Barreto S. L. . 2005 . Brazilian Tables for Poultry and Swine: Composition of Feedstuffs and Nutritional Requirements . Universidade Federal de Viçosa, Departamento de Zootecnia , MG, Brazil . 11. Quantum Blue 5 G (AB Vista, Marlborough UK) with an expected activity of 5,000 FTU/g . 12. Econase XT 25 G (AB Vista, Marlborough UK) with an expected activity of 160,000 BXU/g . 13. ProAct (DSM, Heerlen, the Netherlands) with a minimum activity of 75,000 PROT/g. 14. Engelen A. J. , van der Heeft F. C. , Randsdorp P. H. G. , Somers W. A. C. . 2001 . Determination of phytase activity in feed by colorimetric enzymatic method: Collaborative interlaboratory study . J. AOAC. Int. 84 : 629 – 633 . Briefly samples were prepared and analyzed for phytase activity using phytic acid from rice as a substrate (extraction for 60 minutes in 250 mM acetate pH 5.5 etc. buffer, analysis at pH 5.5 and 37°C). The method is based on the end-point determination of phosphate using a molybdate-vanadate colour system. The colour produced is proportional to enzyme activity. Units are measured as FTU/g. Google Scholar PubMed 15. Samples were analyzed for xylanase activity using birch xylan as a substrate at pH 5.3 and 50°C. The method is based on the end-point determination of reducing sugars using a DNS-based colorimetric system. The colour produced is proportional to enzyme activity. Units are expressed as nanomoles per second of xylose reducing sugar equivalents (BXU) . 16. Samples were analyzed for neutral protease activity using casein as a substrate at pH 7.5 and 50°C. Amino acid determination is carried out on the filtrate/supernatant using Folin's reagent which serves as a phenol colour developer. The quantity of amino acid released is proportional to enzyme activity in the original sample. 17. Association of Official Analytical Chemists . 1951 . Calcium in Animal Feed method 935.13 in Official Methods of Analysis . Assoc. Anal. Chem. , Washington, DC . 18. Association of Official Analytical Chemists . 1970 . Animal Feeds. Phosphorus method 7.095-7.098 in Official Methods of Analysis . Assoc. Anal. Chem. , Washington, DC . 19. Association of Official Analytical Chemists . 2001 . Protein in Animal Feed, Forage, Grain and Oilseeds. 990.03 in Official Methods of Analysis . Assoc. Anal. Chem. , Washington, DC . 20. JMP Pro . 2016 . Version 13.0.0 ed . SAS Institute, Inc. Cary, NC . 21. Protease units are defined as the amount of protease that liberates 1 umol para-nitroaniline (pNA) from 1 mM Suc-Ala-Ala-Pro-Phe-pNa per minute at pH 9 and 37°C . 22. Yuan L. , Wang M. , Zhang X. , Wang Z. . 2017 . Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers . PLoS One . 12 : e0173941 . Google Scholar CrossRef Search ADS PubMed 23. Walk C. L. , Cowieson A. J. , Remus J. C. , Novak C. L. , McElroy A. P. . 2011 . Effects of dietary enzymes on performance and intestinal goblet cell number of broilers exposed to a live coccidia oocyst vaccine . Poult. Sci. 90 : 91 – 98 . Google Scholar CrossRef Search ADS PubMed 24. Schramm V. G. , Durau J. F. , Barrilli L. N. E. , Sorbara J. O. B. , Cowieson A. J. , Feliz A. P. , Maiorka A. . 2017 . Interaction between xylanase and phytase on the digestibility of corn and a corn/soy diet for broiler chickens . Poult. Sci. 96 : 1204 – 1211 . Google Scholar PubMed 25. Cowieson A. J. , Roos F. F. . 2014 . Bioefficacy of a mono-component protease in the diets of pigs and poultry: a meta-analysis of effect on ileal amino acid digestibility . J. Appl. Anim. Nutr. 2 : 1 – 8 . Google Scholar CrossRef Search ADS 26. Beaulac K. R. , Bedford M. R. , van Kessel A. , Schwean-Lardner K. , Kautzman M. E. , Abbott D. A. , Classen H. L. . 2015 . High levels of dietary phytase increase growth and nutrient digestibility in young broilers . Poult. Sci. 94 ( E-Suppl.1 ): 32 . 27. Masey O’Neill V. H. , Smith J. A. , Bedford M. R. . 2014 . Multicarbohydrase enzymes for non-ruminants . Asian Australas. J. Anim. Sci 27 : 290 – 301 . Google Scholar CrossRef Search ADS PubMed 28. Santos dos T. T. , Masey O’Neill H. V. , Gonzalez-Ortiz G. , Camacho-Fernandez D. , Lopez-Coello C. . 2017 . Xylanase, protease and superdosing phytase interactions in broiler performance, carcass yield and digesta transit time . Anim. Nutr. 3 : 121 – 126 . Google Scholar CrossRef Search ADS 29. Lee S. , Bedford M. R. , Walk C. L. . 2017 . Meta-analysis: explicit value of mono-component proteases in monogastric diets . Poult. Sci. http://dx.doi.org/10.3382/ps/pey042 (accessed on 18 April 2018) . © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Poultry Research Oxford University Press

Evaluation of Phytase, Xylanase, and Protease in Reduced Nutrient Diets Fed to Broilers

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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1056-6171
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1537-0437
D.O.I.
10.3382/japr/pfy022
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Abstract

Abstract Two experiments were conducted to evaluate phytase, phytase + xylanase, or phytase + xylanase + protease in diets deficient in Ca, available phosphorus (avP), energy, and amino acids. Lohman Indian River, straight-run broilers (n = 2,304 and 4,752 in experiments 1 and 2, respectively) were fed one of eight diets: (1) nutrient adequate control (PC), (2) negative control (NC) 1 with reduced Ca and avP, (3) NC1 + 1,500 FTU/kg phytase, (4) NC2 with reduced Ca, avP and energy, (5) NC2 + 1,500 FTU/kg phytase + 16,000 BXU/kg xylanase, (6) NC3 with reduced Ca, avP, energy, and amino acids, (7) NC3 + 1,500 FTU/kg phytase + 16,000 BXU/kg xylanase, (8) NC3 + 1,500 FTU/kg phytase + 16,000 BXU/kg xylanase + 15,000 U/kg protease. Reducing Ca, avP, energy, and amino acids significantly reduced body weight gain (BWG) and feed efficiency in a sequential fashion, with birds fed NC3 having a significantly higher FCR compared with birds fed NC1 or the PC. In both experiments, supplementation with phytase, phytase + xylanase, or the combination of phytase + xylanase + protease significantly improved BWG and FCR comparable to the PC. However, there was no difference in BWG or FCR between birds fed phytase + xylanase or phytase + xylanase + protease. Results indicate that supplementation of phytase + xylanase restored the BWG and FCR of the birds fed reduced Ca, avP, energy, and amino acid diets, and supplementation of protease to diets containing phytase and xylanase needs further investigation. DESCRIPTION OF PROBLEM The use of exogenous enzymes, specifically phytase and xylanase, is reported to save the global feed market an estimated 3–5 billion dollars per year [1]. Numerous research trials have evaluated the influence of enzymes individually or in combination on the growth performance and nutrient digestibility of broiler chicks. In most cases, the enzymes are applied to diets containing marginal levels of Ca, available phosphorus (avP), and energy, particularly when phytase, xylanase, amylase + protease, phytase + xylanase, or xylanase + protease are evaluated [2, 3, 4]. It is believed that each enzyme works independently to provide specific nutrients to the bird in an additive or sub-additive fashion. For example, protease is reported to increase amino acid digestibility, as well as have ancillary effects on starch, fat and digestible energy with inconsistent effects on growth performance as reviewed by Cowieson and Roos [5]. Supplementation of xylanase into corn-based diets is reported to increase fat and starch digestibility through improved digestion of resistant starch, improved access to cell walls via reduction in cell wall integrity, and/or modification of intestinal microbial communities through the production of prebiotic-like oligosaccharides [2]. Carbohydrases, such as xylanase, are also reported to increase apparent amino acid digestibility over the control diet by approximately 4.5%, which is reduced to 4.0% in the presence of phytase [6]. Phytase is known to hydrolyze phytate P in plants, thereby increasing the avP content of the diet and enabling reductions in added inorganic P, while at the same time reducing the anti-nutrient effects of phytate. This antinutritive effect extends to minerals, specifically Ca, allowing for reductions in Ca concentrations in the diet, and to energy and amino acids as reviewed by Dersjant-Li et al. [7]. However, the effect of phytase on energy and amino acid digestibility is less consistent than the effects of phytase on P. For example, Cowieson and Adeola [3] reported phytase had no effect on ileal digestible energy in broilers fed diets deficient in Ca, P, and energy (180 kcal/kg). In that same trial, performance tended to improve with phytase supplementation or supplementation of xylanase + amylase + protease, with the greatest performance achieved in the deficient diets when phytase was supplemented in combination with xylanase + amylase + protease. Questions remain regarding the benefits of supplementing broiler diets, marginal in Ca, avP, energy, and amino acids with phytase alone, or phytase + carbohydrases, or phytase + carbohydrase + protease to provide additional nutrients over and above that of a combination of phytase and carbohydrase. Therefore, the objective of this set of trials was to determine the effect of supplemental phytase alone and in combination with xylanase and protease on broiler performance when fed diets with reductions in Ca, avP, energy, and amino acids. MATERIALS AND METHODS The trial was conducted at Japfa Poultry Research Facilities under the guidelines of the ethical principles for use of animals for scientific purposes. Experiment 1 Straight-run broilers (n = 2,304) were obtained at hatch and housed in 128 battery cages in a closed house with tunnel ventilation [8]. There were 18 birds per cage and 16 replicate cages per treatment. Birds had ad libitum access to feed and water throughout the 21-d study. Starter diets were fed in crumble form. Birds and feed were weighed at the start (d 0) and end (d 21) of the experiment to determine feed intake (FI) and body weight gain (BWG) and calculate FCR. Culled or dead birds were recorded and weighed and FI was adjusted for bird days. Experiment 2 Straight-run broilers (n = 4,752) were obtained at hatch and housed in 72 floor pens [9]. There were 66 birds per pen and 9 replicate pens per treatment. Birds had ad libitum access to feed and water provided by bell feeders and drinkers for the 35-d study. Birds were fed crumble starter diets from hatch to 21 d and pelleted grower diets from 22 to 35 d post-hatch. Birds and feed were weighed at the start (d 0), d 21, and end (d 35) of the experiment to determine FI and BWG and calculate FCR. Culled or dead birds were recorded and weighed and FI was adjusted for bird days. Experimental Design and Statistics Four starter (experiments 1 and 2) and grower (experiment 2) basal diets (Tables 1 and 2) were mixed: (1) a nutrient adequate PC formulated to meet or exceed Rostagno et al. [10] nutrient requirements for 21 and 35-d-old broilers; (2) negative control (NC) 1, reduced in Ca by 0.20% and avP by 0.18% compared with the PC; (3) NC2, reduced in Ca by 0.20%, avP by 0.18%, and ME by 80 kcal/kg compared with the PC; and (4) NC3, reduced in Ca by 0.20%, avP by 0.18%, ME by 80 kcal/kg, and digestible amino acids by 5% compared with the PC. The remaining four experimental diets were as follows: (5) NC1 plus 1,500 FTU/kg phytase [11]; (6) NC2 plus 1,500 FTU/kg and 16,000 BXU/kg xylanase [12]; (7) NC3 plus 1,500 FTU/kg and 16,000 BXU/kg; and (8) NC3 plus 1,500 FTU/kg, 16,000 BXU/kg, and 15,000 U/kg protease [13]. Table 1. Ingredient and nutrient composition of the starter diets (as is basis). Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 55.28 54.46 53.81 55.43 Corn gluten meal 3.78 3.79 3.80 3.12 Corn dried distillers grains with solubles 4.50 4.50 4.50 4.50 Wheat bran 3.22 5.56 7.64 8.54 Soybean meal 25.94 25.45 25.01 23.16 Palm oil 2.80 2.80 1.80 1.80 Limestone 1.58 1.39 1.40 1.41 Monocalcium phosphate 1.36 0.50 0.49 0.50 Salt 0.23 0.23 0.23 0.23 L—lysine HCl 0.35 0.36 0.36 0.35 DL—methionine 0.25 0.25 0.25 0.23 L—threonine 0.09 0.09 0.10 0.09 Sodium bicarbonate 0.30 0.30 0.31 0.30 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.04 0.04 0.04 0.04 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.72 88.64 88.55 88.52 Fat 5.75 5.80 4.86 4.93 Fiber 2.65 2.84 3.02 3.05 Ash 5.99 5.20 5.26 5.21 Crude protein 21.33 21.38 21.43 20.40 ME, kcal/kg 3,000.00 3,000.00 2,920.00 2,920.00 Digestible lysine 1.15 1.15 1.15 1.09 Digestible methionine 0.57 0.57 0.56 0.53 Digestible TSAA 0.85 0.85 0.85 0.81 Digestible tryptophan 0.20 0.20 0.21 0.20 Digestible arginine 1.21 1.21 1.21 1.15 Digestible threonine 0.76 0.76 0.76 0.72 Digestible leucine 1.84 1.83 1.82 1.72 Digestible valine 0.88 0.88 0.87 0.83 Digestible isoleucine 0.78 0.78 0.78 0.73 Calcium 0.90 0.70 0.70 0.70 Total phosphorus 0.70 0.53 0.54 0.54 Available phosphorus 0.42 0.24 0.24 0.24 Sodium 0.19 0.19 0.19 0.19 Chloride 0.25 0.25 0.25 0.25 Choline 1,400.00 1,400.00 1,400.00 1,400.00 Analyzed nutrient composition, % Crude protein 21.4 21.4 21.5 20.07 Calcium 0.84 0.71 0.68 0.69 Total phosphorus 0.69 0.54 0.54 0.55 Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 55.28 54.46 53.81 55.43 Corn gluten meal 3.78 3.79 3.80 3.12 Corn dried distillers grains with solubles 4.50 4.50 4.50 4.50 Wheat bran 3.22 5.56 7.64 8.54 Soybean meal 25.94 25.45 25.01 23.16 Palm oil 2.80 2.80 1.80 1.80 Limestone 1.58 1.39 1.40 1.41 Monocalcium phosphate 1.36 0.50 0.49 0.50 Salt 0.23 0.23 0.23 0.23 L—lysine HCl 0.35 0.36 0.36 0.35 DL—methionine 0.25 0.25 0.25 0.23 L—threonine 0.09 0.09 0.10 0.09 Sodium bicarbonate 0.30 0.30 0.31 0.30 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.04 0.04 0.04 0.04 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.72 88.64 88.55 88.52 Fat 5.75 5.80 4.86 4.93 Fiber 2.65 2.84 3.02 3.05 Ash 5.99 5.20 5.26 5.21 Crude protein 21.33 21.38 21.43 20.40 ME, kcal/kg 3,000.00 3,000.00 2,920.00 2,920.00 Digestible lysine 1.15 1.15 1.15 1.09 Digestible methionine 0.57 0.57 0.56 0.53 Digestible TSAA 0.85 0.85 0.85 0.81 Digestible tryptophan 0.20 0.20 0.21 0.20 Digestible arginine 1.21 1.21 1.21 1.15 Digestible threonine 0.76 0.76 0.76 0.72 Digestible leucine 1.84 1.83 1.82 1.72 Digestible valine 0.88 0.88 0.87 0.83 Digestible isoleucine 0.78 0.78 0.78 0.73 Calcium 0.90 0.70 0.70 0.70 Total phosphorus 0.70 0.53 0.54 0.54 Available phosphorus 0.42 0.24 0.24 0.24 Sodium 0.19 0.19 0.19 0.19 Chloride 0.25 0.25 0.25 0.25 Choline 1,400.00 1,400.00 1,400.00 1,400.00 Analyzed nutrient composition, % Crude protein 21.4 21.4 21.5 20.07 Calcium 0.84 0.71 0.68 0.69 Total phosphorus 0.69 0.54 0.54 0.55 1Trace mineral premix provided per kg of diet: selenium 0.09 mg, iodine 0.18 mg, copper 3.00 mg, iron 36.0 mg, manganese 54.0 mg, zinc 48.0 mg, cobalt 0.12 mg. 2Vitamin premix provided per kg of diet: Vit A 11,550 IU, Vit D3 4,300 IU, Vit E 27.5 IU, Vit K3 3.85 mg, Vit B1 2.75 mg, Vit B2 9.9 mg, Vit B6 3.85 mg, Vit B12 22.0 Mcg, niacin 49.5 mg, pantothenic acid 15.4 mg, folic acid 1.38 mg, biotin 166 Mcg, selenium 0.08 mg, iodine 1.38 mg. View Large Table 1. Ingredient and nutrient composition of the starter diets (as is basis). Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 55.28 54.46 53.81 55.43 Corn gluten meal 3.78 3.79 3.80 3.12 Corn dried distillers grains with solubles 4.50 4.50 4.50 4.50 Wheat bran 3.22 5.56 7.64 8.54 Soybean meal 25.94 25.45 25.01 23.16 Palm oil 2.80 2.80 1.80 1.80 Limestone 1.58 1.39 1.40 1.41 Monocalcium phosphate 1.36 0.50 0.49 0.50 Salt 0.23 0.23 0.23 0.23 L—lysine HCl 0.35 0.36 0.36 0.35 DL—methionine 0.25 0.25 0.25 0.23 L—threonine 0.09 0.09 0.10 0.09 Sodium bicarbonate 0.30 0.30 0.31 0.30 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.04 0.04 0.04 0.04 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.72 88.64 88.55 88.52 Fat 5.75 5.80 4.86 4.93 Fiber 2.65 2.84 3.02 3.05 Ash 5.99 5.20 5.26 5.21 Crude protein 21.33 21.38 21.43 20.40 ME, kcal/kg 3,000.00 3,000.00 2,920.00 2,920.00 Digestible lysine 1.15 1.15 1.15 1.09 Digestible methionine 0.57 0.57 0.56 0.53 Digestible TSAA 0.85 0.85 0.85 0.81 Digestible tryptophan 0.20 0.20 0.21 0.20 Digestible arginine 1.21 1.21 1.21 1.15 Digestible threonine 0.76 0.76 0.76 0.72 Digestible leucine 1.84 1.83 1.82 1.72 Digestible valine 0.88 0.88 0.87 0.83 Digestible isoleucine 0.78 0.78 0.78 0.73 Calcium 0.90 0.70 0.70 0.70 Total phosphorus 0.70 0.53 0.54 0.54 Available phosphorus 0.42 0.24 0.24 0.24 Sodium 0.19 0.19 0.19 0.19 Chloride 0.25 0.25 0.25 0.25 Choline 1,400.00 1,400.00 1,400.00 1,400.00 Analyzed nutrient composition, % Crude protein 21.4 21.4 21.5 20.07 Calcium 0.84 0.71 0.68 0.69 Total phosphorus 0.69 0.54 0.54 0.55 Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 55.28 54.46 53.81 55.43 Corn gluten meal 3.78 3.79 3.80 3.12 Corn dried distillers grains with solubles 4.50 4.50 4.50 4.50 Wheat bran 3.22 5.56 7.64 8.54 Soybean meal 25.94 25.45 25.01 23.16 Palm oil 2.80 2.80 1.80 1.80 Limestone 1.58 1.39 1.40 1.41 Monocalcium phosphate 1.36 0.50 0.49 0.50 Salt 0.23 0.23 0.23 0.23 L—lysine HCl 0.35 0.36 0.36 0.35 DL—methionine 0.25 0.25 0.25 0.23 L—threonine 0.09 0.09 0.10 0.09 Sodium bicarbonate 0.30 0.30 0.31 0.30 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.04 0.04 0.04 0.04 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.72 88.64 88.55 88.52 Fat 5.75 5.80 4.86 4.93 Fiber 2.65 2.84 3.02 3.05 Ash 5.99 5.20 5.26 5.21 Crude protein 21.33 21.38 21.43 20.40 ME, kcal/kg 3,000.00 3,000.00 2,920.00 2,920.00 Digestible lysine 1.15 1.15 1.15 1.09 Digestible methionine 0.57 0.57 0.56 0.53 Digestible TSAA 0.85 0.85 0.85 0.81 Digestible tryptophan 0.20 0.20 0.21 0.20 Digestible arginine 1.21 1.21 1.21 1.15 Digestible threonine 0.76 0.76 0.76 0.72 Digestible leucine 1.84 1.83 1.82 1.72 Digestible valine 0.88 0.88 0.87 0.83 Digestible isoleucine 0.78 0.78 0.78 0.73 Calcium 0.90 0.70 0.70 0.70 Total phosphorus 0.70 0.53 0.54 0.54 Available phosphorus 0.42 0.24 0.24 0.24 Sodium 0.19 0.19 0.19 0.19 Chloride 0.25 0.25 0.25 0.25 Choline 1,400.00 1,400.00 1,400.00 1,400.00 Analyzed nutrient composition, % Crude protein 21.4 21.4 21.5 20.07 Calcium 0.84 0.71 0.68 0.69 Total phosphorus 0.69 0.54 0.54 0.55 1Trace mineral premix provided per kg of diet: selenium 0.09 mg, iodine 0.18 mg, copper 3.00 mg, iron 36.0 mg, manganese 54.0 mg, zinc 48.0 mg, cobalt 0.12 mg. 2Vitamin premix provided per kg of diet: Vit A 11,550 IU, Vit D3 4,300 IU, Vit E 27.5 IU, Vit K3 3.85 mg, Vit B1 2.75 mg, Vit B2 9.9 mg, Vit B6 3.85 mg, Vit B12 22.0 Mcg, niacin 49.5 mg, pantothenic acid 15.4 mg, folic acid 1.38 mg, biotin 166 Mcg, selenium 0.08 mg, iodine 1.38 mg. View Large Table 2. Ingredient and nutrient composition of the grower diets (as is basis). Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 62.05 61.23 60.59 62.05 Corn gluten meal 3.60 3.61 3.61 2.93 Corn dried distillers grains with solubles 6.00 6.00 6.00 6.00 Wheat bran 3.02 5.35 7.43 8.12 Soybean meal 18.60 18.11 17.67 16.20 Palm oil 3.00 3.00 2.00 2.00 Limestone 1.38 1.19 1.20 1.20 Monocalcium phosphate 1.02 0.16 0.15 0.16 Salt 0.18 0.18 0.18 0.18 L—lysine HCl 0.32 0.33 0.33 0.32 DL—methionine 0.17 0.17 0.17 0.16 L—threonine 0.07 0.07 0.07 0.07 Sodium bicarbonate 0.28 0.29 0.29 0.29 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.05 0.05 0.04 0.05 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.62 88.53 88.45 88.41 Fat 6.22 6.28 5.34 5.40 Fiber 2.56 2.75 2.92 2.95 Ash 5.12 4.33 4.40 4.35 Crude protein 18.54 18.59 18.64 17.75 ME, kcal/kg 3,100.00 3,100.00 3,020.00 3,020.00 Digestible lysine 0.95 0.95 0.95 0.90 Digestible methionine 0.46 0.46 0.46 0.44 Digestible TSAA 0.72 0.72 0.72 0.69 Digestible tryptophan 0.17 0.17 0.17 0.16 Digestible arginine 1.00 1.00 1.00 0.95 Digestible threonine 0.64 0.64 0.64 0.61 Digestible valine 0.76 0.76 0.76 0.72 Digestible leucine 1.67 1.66 1.66 1.56 Digestible isoleucine 0.66 0.66 0.65 0.61 Calcium 0.75 0.55 0.55 0.55 Total phosphorus 0.60 0.43 0.44 0.44 Available phosphorus 0.34 0.16 0.16 0.16 Sodium 0.17 0.17 0.17 0.17 Chloride 0.22 0.22 0.22 0.22 Choline 1,300.00 1,300.00 1,300.00 1,300.00 Analyzed nutrient composition, % Crude protein 18.3 18.9 18.6 17.7 Calcium 0.67 0.54 0.54 0.56 Total phosphorus 0.58 0.44 0.45 0.44 Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 62.05 61.23 60.59 62.05 Corn gluten meal 3.60 3.61 3.61 2.93 Corn dried distillers grains with solubles 6.00 6.00 6.00 6.00 Wheat bran 3.02 5.35 7.43 8.12 Soybean meal 18.60 18.11 17.67 16.20 Palm oil 3.00 3.00 2.00 2.00 Limestone 1.38 1.19 1.20 1.20 Monocalcium phosphate 1.02 0.16 0.15 0.16 Salt 0.18 0.18 0.18 0.18 L—lysine HCl 0.32 0.33 0.33 0.32 DL—methionine 0.17 0.17 0.17 0.16 L—threonine 0.07 0.07 0.07 0.07 Sodium bicarbonate 0.28 0.29 0.29 0.29 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.05 0.05 0.04 0.05 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.62 88.53 88.45 88.41 Fat 6.22 6.28 5.34 5.40 Fiber 2.56 2.75 2.92 2.95 Ash 5.12 4.33 4.40 4.35 Crude protein 18.54 18.59 18.64 17.75 ME, kcal/kg 3,100.00 3,100.00 3,020.00 3,020.00 Digestible lysine 0.95 0.95 0.95 0.90 Digestible methionine 0.46 0.46 0.46 0.44 Digestible TSAA 0.72 0.72 0.72 0.69 Digestible tryptophan 0.17 0.17 0.17 0.16 Digestible arginine 1.00 1.00 1.00 0.95 Digestible threonine 0.64 0.64 0.64 0.61 Digestible valine 0.76 0.76 0.76 0.72 Digestible leucine 1.67 1.66 1.66 1.56 Digestible isoleucine 0.66 0.66 0.65 0.61 Calcium 0.75 0.55 0.55 0.55 Total phosphorus 0.60 0.43 0.44 0.44 Available phosphorus 0.34 0.16 0.16 0.16 Sodium 0.17 0.17 0.17 0.17 Chloride 0.22 0.22 0.22 0.22 Choline 1,300.00 1,300.00 1,300.00 1,300.00 Analyzed nutrient composition, % Crude protein 18.3 18.9 18.6 17.7 Calcium 0.67 0.54 0.54 0.56 Total phosphorus 0.58 0.44 0.45 0.44 1Trace mineral premix provided per kg of diet: selenium 0.09 mg, iodine 0.18 mg, copper 3.00 mg, iron 36.0 mg, manganese 54.0 mg, zinc 48.0 mg, cobalt 0.12 mg. 2Vitamin premix provided per kg of diet: Vit A 9,450 IU, Vit D3 3,700 IU, Vit E 22.5 IU, Vit K3 3.15 mg, Vit B1 2.25 mg, Vit B2 8.1 mg, Vit B6 3.15 mg, Vit B12 18.0 Mcg, niacin 40.5 mg, pantothenic acid 12.6 mg, folic acid 1.13 mg, biotin 116 Mcg, selenium 0.07 mg, iodine 1.13 mg. View Large Table 2. Ingredient and nutrient composition of the grower diets (as is basis). Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 62.05 61.23 60.59 62.05 Corn gluten meal 3.60 3.61 3.61 2.93 Corn dried distillers grains with solubles 6.00 6.00 6.00 6.00 Wheat bran 3.02 5.35 7.43 8.12 Soybean meal 18.60 18.11 17.67 16.20 Palm oil 3.00 3.00 2.00 2.00 Limestone 1.38 1.19 1.20 1.20 Monocalcium phosphate 1.02 0.16 0.15 0.16 Salt 0.18 0.18 0.18 0.18 L—lysine HCl 0.32 0.33 0.33 0.32 DL—methionine 0.17 0.17 0.17 0.16 L—threonine 0.07 0.07 0.07 0.07 Sodium bicarbonate 0.28 0.29 0.29 0.29 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.05 0.05 0.04 0.05 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.62 88.53 88.45 88.41 Fat 6.22 6.28 5.34 5.40 Fiber 2.56 2.75 2.92 2.95 Ash 5.12 4.33 4.40 4.35 Crude protein 18.54 18.59 18.64 17.75 ME, kcal/kg 3,100.00 3,100.00 3,020.00 3,020.00 Digestible lysine 0.95 0.95 0.95 0.90 Digestible methionine 0.46 0.46 0.46 0.44 Digestible TSAA 0.72 0.72 0.72 0.69 Digestible tryptophan 0.17 0.17 0.17 0.16 Digestible arginine 1.00 1.00 1.00 0.95 Digestible threonine 0.64 0.64 0.64 0.61 Digestible valine 0.76 0.76 0.76 0.72 Digestible leucine 1.67 1.66 1.66 1.56 Digestible isoleucine 0.66 0.66 0.65 0.61 Calcium 0.75 0.55 0.55 0.55 Total phosphorus 0.60 0.43 0.44 0.44 Available phosphorus 0.34 0.16 0.16 0.16 Sodium 0.17 0.17 0.17 0.17 Chloride 0.22 0.22 0.22 0.22 Choline 1,300.00 1,300.00 1,300.00 1,300.00 Analyzed nutrient composition, % Crude protein 18.3 18.9 18.6 17.7 Calcium 0.67 0.54 0.54 0.56 Total phosphorus 0.58 0.44 0.45 0.44 Ingredient, % Positive control Negative control 1 Negative control 2 Negative control 3 Corn 62.05 61.23 60.59 62.05 Corn gluten meal 3.60 3.61 3.61 2.93 Corn dried distillers grains with solubles 6.00 6.00 6.00 6.00 Wheat bran 3.02 5.35 7.43 8.12 Soybean meal 18.60 18.11 17.67 16.20 Palm oil 3.00 3.00 2.00 2.00 Limestone 1.38 1.19 1.20 1.20 Monocalcium phosphate 1.02 0.16 0.15 0.16 Salt 0.18 0.18 0.18 0.18 L—lysine HCl 0.32 0.33 0.33 0.32 DL—methionine 0.17 0.17 0.17 0.16 L—threonine 0.07 0.07 0.07 0.07 Sodium bicarbonate 0.28 0.29 0.29 0.29 Mineral premix1 0.03 0.03 0.03 0.03 Choline chloride 0.05 0.05 0.04 0.05 Vitamin premix2 0.25 0.25 0.25 0.25 Formulated nutrient composition, % Dry matter 88.62 88.53 88.45 88.41 Fat 6.22 6.28 5.34 5.40 Fiber 2.56 2.75 2.92 2.95 Ash 5.12 4.33 4.40 4.35 Crude protein 18.54 18.59 18.64 17.75 ME, kcal/kg 3,100.00 3,100.00 3,020.00 3,020.00 Digestible lysine 0.95 0.95 0.95 0.90 Digestible methionine 0.46 0.46 0.46 0.44 Digestible TSAA 0.72 0.72 0.72 0.69 Digestible tryptophan 0.17 0.17 0.17 0.16 Digestible arginine 1.00 1.00 1.00 0.95 Digestible threonine 0.64 0.64 0.64 0.61 Digestible valine 0.76 0.76 0.76 0.72 Digestible leucine 1.67 1.66 1.66 1.56 Digestible isoleucine 0.66 0.66 0.65 0.61 Calcium 0.75 0.55 0.55 0.55 Total phosphorus 0.60 0.43 0.44 0.44 Available phosphorus 0.34 0.16 0.16 0.16 Sodium 0.17 0.17 0.17 0.17 Chloride 0.22 0.22 0.22 0.22 Choline 1,300.00 1,300.00 1,300.00 1,300.00 Analyzed nutrient composition, % Crude protein 18.3 18.9 18.6 17.7 Calcium 0.67 0.54 0.54 0.56 Total phosphorus 0.58 0.44 0.45 0.44 1Trace mineral premix provided per kg of diet: selenium 0.09 mg, iodine 0.18 mg, copper 3.00 mg, iron 36.0 mg, manganese 54.0 mg, zinc 48.0 mg, cobalt 0.12 mg. 2Vitamin premix provided per kg of diet: Vit A 9,450 IU, Vit D3 3,700 IU, Vit E 22.5 IU, Vit K3 3.15 mg, Vit B1 2.25 mg, Vit B2 8.1 mg, Vit B6 3.15 mg, Vit B12 18.0 Mcg, niacin 40.5 mg, pantothenic acid 12.6 mg, folic acid 1.13 mg, biotin 116 Mcg, selenium 0.07 mg, iodine 1.13 mg. View Large Phytase recoveries in the diets were analyzed using a modified method of Engelen et al. [14]. Xylanase recovery in the experimental diets were analyzed using in house methods [15] and protease activity was analyzed using casein as a substrate [16]. Experimental diets were also analyzed for Ca [17] and total P [18], and crude protein [19]. Finally, data were arranged as a completely randomized design and analyzed as a 1-way ANOVA using the fit model platform in JMP Pro [20]. The model included diet. Pen served as the experimental unit. Data are presented as least square means per treatment group. Significant differences between the means were separated using multiple t-tests and significance was accepted at P < 0.05. RESULTS AND DISCUSSION Experimental Diets Nutrient analyses in the diets were similar to formulated (Table 1). However, phytase recoveries in the diets containing phytase (expected 1,500 FTU/kg) were approximately 50% lower than expected at 965, 698, 543, and 706 FTU/kg in the starter diets and 980, 862, 1,150, and 867 FTU/kg in the grower diets. The phytase activity of the premix analyzed as expected, at 6,590 FTU/g, when overages in the product from the manufacturer are considered. Xylanase recovery in the diets containing xylanase was approximately 17% higher than formulated and this is within the expected ranges from the manufacturer. Protease recovery in the diets was not performed due to lack of a suitable in feed assay. However, protease activity in the enzyme sample was analyzed at 4,42,000 U/g as described previously [16]. The expected activity of the protease was 75,000 protease U/g based on manufacturer assay recommendations [21], which were different from the assay used to determine protease activity in this experiment and may explain the differences reported between this paper and the manufacturer expectations. Experiment 1 Overall mortality was low (2.43%) and not associated (P = 0.81) with experimental diets (Table 3). Feed intake and BWG were reduced (P < 0.0001) in birds fed the NC diets compared with birds fed all other diets (Table 3). This has been previously reported in corn-soy diets fed to broilers marginally deficient in energy and essential amino acids [2], and in corn-soy diets containing wheat or rye and deficient in Ca, non-phytate P, and ME [3,4]. Supplementation of the NC2 or NC3 with phytase + xylanase or phytase + xylanase + protease increased (P < 0.0001) FI and BWG above that of birds fed the NCs and comparable to birds fed the PC (Table 3). Body weight gain was significantly higher in birds fed NC1 + 1,500 FTU/kg phytase compared with birds fed the PC (Table 3). In addition, supplementation of NC1 with 1,500 FTU/kg of phytase improved FCR compared with all other diets, except birds fed NC2 with phytase + xylanase, and this was comparable to the PC (Table 3). Feed conversion ratio was highest (P < 0.0001) in birds fed NC3 without any enzyme supplementation compared to all other diets, confirming the reduction in Ca, avP, energy, and amino acids significantly impacted FCR (Table 3). Supplementation of NC3 with phytase + xylanase significantly improved FCR and this was comparable to the PC, NC1, or NC2. Addition of protease to the phytase and xylanase combination had no additional benefit on FCR. Yuan et al. [22] reported significant improvements in FCR of 21-d-old broilers fed nutrient adequate corn-soy diets supplemented with carbohydrases (xylanase, beta-glucanase, and cellulase) with no further improvements in FCR with the addition of various doses of an added protease. Others have reported significantly improved BWG and FCR of 21-d-old broilers fed xylanase, protease, and amylase supplemented corn-soy diets whether they were formulated to be nutrient adequate or marginally deficient in energy and essential amino acids [2]. However, the enzymes in the previous trial were not evaluated individually or in combination with a phytase, and others have reported 21-d-old broilers fed corn, wheat, soy diets deficient in Ca, non-phytate P and ME gained more and were more efficient when the diets were supplemented with 1,000 FTU/kg of phytase regardless of the inclusion of an enzyme cocktail containing xylanase, amylase, or protease [4]. Table 3. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 21 d post-hatch (experiment 1) Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,301.3a 992.7b 1.299b,c 1.70 Negative control 12 0 0 0 1,185.8b 898.5c 1.311b 2.80 1,500 0 0 1,311.7a 1017.6a 1.269d 2.10 Negative control 23 0 0 0 1,178.2b 887.3c 1.314b 1.70 1,500 16,000 0 1,302.9a 1,000.3a,b 1.284c,d 3.10 Negative control 34 0 0 0 1,203.2b 892.2c 1.337a 3.50 1,500 16,000 0 1,311.4a 1,009.2a,b 1.304b 1.40 1,500 16,000 15,000 1,330.2a 1,010.5a,b 1.304b 3.10 SEM 12.0 8.2 0.01 1.1 P-value <0.0001 <0.0001 <0.0001 0.81 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,301.3a 992.7b 1.299b,c 1.70 Negative control 12 0 0 0 1,185.8b 898.5c 1.311b 2.80 1,500 0 0 1,311.7a 1017.6a 1.269d 2.10 Negative control 23 0 0 0 1,178.2b 887.3c 1.314b 1.70 1,500 16,000 0 1,302.9a 1,000.3a,b 1.284c,d 3.10 Negative control 34 0 0 0 1,203.2b 892.2c 1.337a 3.50 1,500 16,000 0 1,311.4a 1,009.2a,b 1.304b 1.40 1,500 16,000 15,000 1,330.2a 1,010.5a,b 1.304b 3.10 SEM 12.0 8.2 0.01 1.1 P-value <0.0001 <0.0001 <0.0001 0.81 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large Table 3. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 21 d post-hatch (experiment 1) Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,301.3a 992.7b 1.299b,c 1.70 Negative control 12 0 0 0 1,185.8b 898.5c 1.311b 2.80 1,500 0 0 1,311.7a 1017.6a 1.269d 2.10 Negative control 23 0 0 0 1,178.2b 887.3c 1.314b 1.70 1,500 16,000 0 1,302.9a 1,000.3a,b 1.284c,d 3.10 Negative control 34 0 0 0 1,203.2b 892.2c 1.337a 3.50 1,500 16,000 0 1,311.4a 1,009.2a,b 1.304b 1.40 1,500 16,000 15,000 1,330.2a 1,010.5a,b 1.304b 3.10 SEM 12.0 8.2 0.01 1.1 P-value <0.0001 <0.0001 <0.0001 0.81 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,301.3a 992.7b 1.299b,c 1.70 Negative control 12 0 0 0 1,185.8b 898.5c 1.311b 2.80 1,500 0 0 1,311.7a 1017.6a 1.269d 2.10 Negative control 23 0 0 0 1,178.2b 887.3c 1.314b 1.70 1,500 16,000 0 1,302.9a 1,000.3a,b 1.284c,d 3.10 Negative control 34 0 0 0 1,203.2b 892.2c 1.337a 3.50 1,500 16,000 0 1,311.4a 1,009.2a,b 1.304b 1.40 1,500 16,000 15,000 1,330.2a 1,010.5a,b 1.304b 3.10 SEM 12.0 8.2 0.01 1.1 P-value <0.0001 <0.0001 <0.0001 0.81 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large Experiment 2 Overall mortality (d 0–35) was 4.22% and not associated (P = 0.17) with diet (Tables 4 and 5). From d 0 to 21 (Table 4) and overall (Table 5), FI was reduced (P < 0.0001) in birds fed the NC diets compared with birds fed all other diets. Body weight gain from d 0 to 21 (Table 4) was lowest in birds fed NC3 compared with birds fed all other diets, except birds fed NC2 and this was comparable to birds fed NC1. Overall (d 0–35), BWG was reduced (P < 0.0001) in birds fed the NC diets compared to birds fed all other diets and enzyme supplementation in the NC diets improved BWG comparable to the PC (Table 5). As reported in experiment 1, FCR from d 0 to 21 was highest (P < 0.0001) in birds fed NC3 without enzyme supplementation compared with birds fed all other diets, except birds fed NC2 and this was similar to birds fed NC1 (Table 4). This suggests that the additional amino acid reduction in the NC diet had a real effect on performance over and above that of Ca, avP, and energy. Birds fed the PC had the best FCR and phytase supplementation in NC1 or phytase + xylanase supplementation in NC2 improved FCR comparable to birds fed the PC. Phytase + xylanase or phytase + xylanase + protease in NC3 improved FCR compared to birds fed NC3 without enzymes, but not comparable to the PC at d 21. However, overall (d 0–35) phytase + xylanase or the combination of phytase + xylanase + protease supplemented in NC3 improved (P < 0.0001) FCR compared with birds fed NC3 without enzymes and comparable to birds fed the PC, NC1 + phytase, or NC2 + phytase + xylanase (Table 5). Birds fed NC3 without enzymes had the highest FCR and this was not different than birds fed NC2 without enzymes, which was not different than birds fed NC1 without enzymes. As mentioned earlier, previous authors have reported significant reductions in performance when birds were fed diets deficient in Ca, avP, energy, and essential amino acids and xylanase + protease + amylase [2] or phytase alone [4] were able to improve growth performance in these low nutrient density diets. Table 4. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 21 d post-hatch (experiment 2) Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,386.8a 1,035.0a,b 1.331d 2.20 Negative control 12 0 0 0 1,232.0b 902.0c 1.361b,c 1.50 1,500 0 0 1,414.5a 1,050.5a 1.339c,d 1.50 Negative control 23 0 0 0 1,209.2b 870.5c,d 1.382a,b 2.40 1,500 16,000 0 1,404.4a 1,033.9a,b 1.357b,c,d 0.50 Negative control 34 0 0 0 1,216.3b 865.1d 1.402a 1.50 1,500 16,000 0 1,385.7a 1,014.5b 1.363b,c 1.20 1,500 16,000 15,000 1,411.2a 1,035.4a,b 1.362b,c 0.70 SEM 14.5 12.0 0.01 0.5 P-value <0.0001 <0.0001 <0.0001 0.11 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,386.8a 1,035.0a,b 1.331d 2.20 Negative control 12 0 0 0 1,232.0b 902.0c 1.361b,c 1.50 1,500 0 0 1,414.5a 1,050.5a 1.339c,d 1.50 Negative control 23 0 0 0 1,209.2b 870.5c,d 1.382a,b 2.40 1,500 16,000 0 1,404.4a 1,033.9a,b 1.357b,c,d 0.50 Negative control 34 0 0 0 1,216.3b 865.1d 1.402a 1.50 1,500 16,000 0 1,385.7a 1,014.5b 1.363b,c 1.20 1,500 16,000 15,000 1,411.2a 1,035.4a,b 1.362b,c 0.70 SEM 14.5 12.0 0.01 0.5 P-value <0.0001 <0.0001 <0.0001 0.11 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20% and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large Table 4. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 21 d post-hatch (experiment 2) Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,386.8a 1,035.0a,b 1.331d 2.20 Negative control 12 0 0 0 1,232.0b 902.0c 1.361b,c 1.50 1,500 0 0 1,414.5a 1,050.5a 1.339c,d 1.50 Negative control 23 0 0 0 1,209.2b 870.5c,d 1.382a,b 2.40 1,500 16,000 0 1,404.4a 1,033.9a,b 1.357b,c,d 0.50 Negative control 34 0 0 0 1,216.3b 865.1d 1.402a 1.50 1,500 16,000 0 1,385.7a 1,014.5b 1.363b,c 1.20 1,500 16,000 15,000 1,411.2a 1,035.4a,b 1.362b,c 0.70 SEM 14.5 12.0 0.01 0.5 P-value <0.0001 <0.0001 <0.0001 0.11 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 1,386.8a 1,035.0a,b 1.331d 2.20 Negative control 12 0 0 0 1,232.0b 902.0c 1.361b,c 1.50 1,500 0 0 1,414.5a 1,050.5a 1.339c,d 1.50 Negative control 23 0 0 0 1,209.2b 870.5c,d 1.382a,b 2.40 1,500 16,000 0 1,404.4a 1,033.9a,b 1.357b,c,d 0.50 Negative control 34 0 0 0 1,216.3b 865.1d 1.402a 1.50 1,500 16,000 0 1,385.7a 1,014.5b 1.363b,c 1.20 1,500 16,000 15,000 1,411.2a 1,035.4a,b 1.362b,c 0.70 SEM 14.5 12.0 0.01 0.5 P-value <0.0001 <0.0001 <0.0001 0.11 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20% and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large Table 5. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 35 d post-hatch (experiment 2). Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 3,710.1a 2,272.4a 1.612d 4.20 Negative control 12 0 0 0 3,498.4b 2,075.8b 1.664b,c 3.70 1,500 0 0 3,810.9a 2,332.0a 1.600d 4.90 Negative control 23 0 0 0 3,447.0b 1,992.1b 1.700a,b 6.20 1,500 16,000 0 3,810.6a 2,306.5a 1.612d 4.00 Negative control 34 0 0 0 3,464.5b 1,991.8b 1.722a 3.90 1,500 16,000 0 3,728.5a 2,263.2a 1.615d 4.20 1,500 16,000 15,000 3,742.4a 2,274.9a 1.625c,d 2.70 SEM 50.6 36.6 0.02 P-value <0.0001 <0.0001 <0.0001 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 3,710.1a 2,272.4a 1.612d 4.20 Negative control 12 0 0 0 3,498.4b 2,075.8b 1.664b,c 3.70 1,500 0 0 3,810.9a 2,332.0a 1.600d 4.90 Negative control 23 0 0 0 3,447.0b 1,992.1b 1.700a,b 6.20 1,500 16,000 0 3,810.6a 2,306.5a 1.612d 4.00 Negative control 34 0 0 0 3,464.5b 1,991.8b 1.722a 3.90 1,500 16,000 0 3,728.5a 2,263.2a 1.615d 4.20 1,500 16,000 15,000 3,742.4a 2,274.9a 1.625c,d 2.70 SEM 50.6 36.6 0.02 P-value <0.0001 <0.0001 <0.0001 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large Table 5. Growth performance of broilers fed reduced nutrient density diets without or with a supplemental phytase, xylanase, and protease from hatch to 35 d post-hatch (experiment 2). Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 3,710.1a 2,272.4a 1.612d 4.20 Negative control 12 0 0 0 3,498.4b 2,075.8b 1.664b,c 3.70 1,500 0 0 3,810.9a 2,332.0a 1.600d 4.90 Negative control 23 0 0 0 3,447.0b 1,992.1b 1.700a,b 6.20 1,500 16,000 0 3,810.6a 2,306.5a 1.612d 4.00 Negative control 34 0 0 0 3,464.5b 1,991.8b 1.722a 3.90 1,500 16,000 0 3,728.5a 2,263.2a 1.615d 4.20 1,500 16,000 15,000 3,742.4a 2,274.9a 1.625c,d 2.70 SEM 50.6 36.6 0.02 P-value <0.0001 <0.0001 <0.0001 Basal diet Phytase, FTU/kg Xylanase, BXU/kg Protease, U/kg Feed intake, g BW gain, g mFCR,1 g:g Mortality, % Positive control (PC) 3,710.1a 2,272.4a 1.612d 4.20 Negative control 12 0 0 0 3,498.4b 2,075.8b 1.664b,c 3.70 1,500 0 0 3,810.9a 2,332.0a 1.600d 4.90 Negative control 23 0 0 0 3,447.0b 1,992.1b 1.700a,b 6.20 1,500 16,000 0 3,810.6a 2,306.5a 1.612d 4.00 Negative control 34 0 0 0 3,464.5b 1,991.8b 1.722a 3.90 1,500 16,000 0 3,728.5a 2,263.2a 1.615d 4.20 1,500 16,000 15,000 3,742.4a 2,274.9a 1.625c,d 2.70 SEM 50.6 36.6 0.02 P-value <0.0001 <0.0001 <0.0001 a–dMeans within a column with different superscripts were significantly different (P < 0.05). 1Feed conversion ratio adjusted for mortality. 2Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively. 3Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, and ME by 80 kcal/kg. 4Diets were deficient in Ca and avP by 0.20 and 0.18%, respectively, ME by 80 kcal/kg, and digestible amino acids by 5%. View Large The BWG or FCR results from two separate trials confirm reducing Ca and avP as well as energy and amino acids was sufficient to reduce BWG and increase FCR in broilers, especially during the starter phase and this continued to market age. Enzyme supplementation in the NCs significantly improved BWG comparable to the PC and this was regardless of the enzymes, the combination, or the NC basal diet. Previous authors have reported carbohydrase (xylanase, beta-glucanase, and cellulase) supplementation in a nutrient adequate diet, with or without the addition of various doses of an acid protease, had no effect on gain of 21-d-old broilers, but resulted in significant improvements in FCR [22]. Reducing energy by 80 kcal in the low Ca and avP diets resulted in further reductions in FCR and supplementation of this diet with phytase + xylanase was able to improve FCR comparable to the PC in both experiments. Previous authors have reported significant improvements in broiler growth performance and/or feed efficiency with phytase [4], xylanase [22], or phytase + xylanase [23] with limited or no improvements in performance reported with the addition of other enzymes. Phytase and xylanase are reported to have sub-additive or additive benefits of in corn soy-based diets [6, 24] as the enzymes may act independently on different substrates. However, their effect on nutrients such as amino acids overlaps and are largely dependent on the digestible fraction or the nutritional value of the control diet to which the enzymes are added [6]. This may also contribute to the lack of an effect with the addition of proteases in the current trial, as the effect of protease is also reported to be dependent on the amino acid digestibility of the control diet [25], which is influenced by phytase dose [6, 26] and with the use of a carbohydrase [6]. Research and interest in protease supplementation in poultry diets has increased substantially in the past 5 years, with benefits in performance and/or amino acid digestibility being reported [5]. However, when considering the benefits of any enzyme supplementation, the response is largely dependent on the digestibility of the control diet. Given the majority of commercial poultry diets contain either a phytase or a carbohydrase and most often a combination of the two; it would be justified to further evaluate the supplementation of proteases in the presence of enzymes commonly used in the field and in a true factorial approach [27]. Recently, Santos et al. [28] tested standard or high doses of phytase, plus or minus xylanase, plus or minus protease in a true factorial design and reported significant improvements in FCR of 21-d-old broilers fed protease, but this effect was lost by day 42 and there was no significant benefit of the enzyme combinations on performance or carcass parameters. Similarly, it is clear from the current study that when the protease was used in a diet containing high doses of phytase and a xylanase, its benefit was not noticed. A recent meta-analysis on protease efficacy in broiler diets indicated only 19% of the protease trials published were conducted in the presence of a phytase and/or carbohydrase [29]. Further work into the benefits of protease in diets containing phytase, particularly at high doses, and a carbohydrase is warranted. CONCLUSIONS AND APPLICATIONS Birds fed diets at reduced levels of Ca, avP, ME, and amino acids had the worst performance, particularly FCR, indicating the reduction of these nutrients was inhibiting optimum growth. Supplementation of the low Ca, avP, ME, and amino acid diet with phytase + xylanase or phytase + xylanase + protease was able to improve BWG and FCR comparable to a nutrient adequate PC diet. There were no differences between the diets supplemented with phytase + xylanase or the diets supplemented with phytase + xylanase + protease. This lack of an effect of protease in a diet containing a phytase and xylanase is assumed to be related to the inherent digestibility of the control diet, which would be high in the presence of a phytase and xylanase. The benefits of supplementing protease enzymes in diets containing phytase and xylanase requires further evaluation and justification in corn soy-based diets with inherently high digestibility. Footnotes Primary Audience: Nutritionists, Formulators, Production Personnel, Scientists REFERENCES 1. Adeola O. , Cowieson A. J. . 2011 . Opportunities and challenges in using exogenous enzymes to improve nonruminant animal production . J. Anim. Sci. 89 : 3189 – 3218 . Google Scholar CrossRef Search ADS PubMed 2. Cowieson A. J. , Ravindran V. . 2008 . Effect of exogenous enzymes in maize-based diets varying in nutrient density for young broilers: growth performance and digestibility of energy, minerals and amino acids . Br. Poult. Sci. 49 : 37 – 44 . Google Scholar CrossRef Search ADS PubMed 3. Cowieson A. J. , Adeloa O. . 2005 . Carbohydrases, protease, and phytase have an additive beneficial effect in nutritionally marginal diets for broiler chicks . Poult. Sci. 84 : 1860 – 1867 . Google Scholar CrossRef Search ADS PubMed 4. Olukosi O. A. , Cowieson A. J. , Adeloa O. . 2008 . Energy utilization and growth performance of broilers receiving diets supplemented with enzymes containing carbohydrase or phytase activity individually or in combination . Br. J. Nutr. 99 : 682 – 690 . Google Scholar CrossRef Search ADS PubMed 5. Cowieson A. J. , Roos F. F. . 2016 . Toward optimal value creation through the application of exogenous mono-component protease in the diets of non-ruminants . Anim. Feed Sci. Tech. 221 : 331 – 340 . Google Scholar CrossRef Search ADS 6. Cowieson A. J. , Bedford M. R. . 2009 . The effect of phytase and carbohydrase on ileal amino acid digestibility in monogastric diets: complimentary mode of action? Worlds Poult. Sci. J. 65 : 609 – 624 . Google Scholar CrossRef Search ADS 7. Dersjant-Li Y. , Awati A. , Schulze H. , Partridge G. . 2015 . Phytase in non-ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors . J. Sci. Food Agric. 95 : 878 – 896 . Google Scholar CrossRef Search ADS PubMed 8. Indian River broilers (Aviagen, Indonesia) were housed in cages of galvanized steel with trough feeders, nipple drinkers, and raised wire floors. The lighting program was designed to provide 23:1 L:D (0-7 days) and 20:4:D (8-35 days). 9. Indian River broilers (Aviagen, Indonesia) were housed in floor pens with new rice hull litter in a closed house with tunnel ventilation The lighting program was designed to provide 23:1 L:D (0-7 days) and 20:4 L:D (8-35 days) . 10. Rostagno H. S. , Teixeira A. , Donzele J. L. , Gomes P. C. , De Oliveira R. F. M. , Lopes D. C. , Ferreira A. J. P. , Toledo Barreto S. L. . 2005 . Brazilian Tables for Poultry and Swine: Composition of Feedstuffs and Nutritional Requirements . Universidade Federal de Viçosa, Departamento de Zootecnia , MG, Brazil . 11. Quantum Blue 5 G (AB Vista, Marlborough UK) with an expected activity of 5,000 FTU/g . 12. Econase XT 25 G (AB Vista, Marlborough UK) with an expected activity of 160,000 BXU/g . 13. ProAct (DSM, Heerlen, the Netherlands) with a minimum activity of 75,000 PROT/g. 14. Engelen A. J. , van der Heeft F. C. , Randsdorp P. H. G. , Somers W. A. C. . 2001 . Determination of phytase activity in feed by colorimetric enzymatic method: Collaborative interlaboratory study . J. AOAC. Int. 84 : 629 – 633 . Briefly samples were prepared and analyzed for phytase activity using phytic acid from rice as a substrate (extraction for 60 minutes in 250 mM acetate pH 5.5 etc. buffer, analysis at pH 5.5 and 37°C). The method is based on the end-point determination of phosphate using a molybdate-vanadate colour system. The colour produced is proportional to enzyme activity. Units are measured as FTU/g. Google Scholar PubMed 15. Samples were analyzed for xylanase activity using birch xylan as a substrate at pH 5.3 and 50°C. The method is based on the end-point determination of reducing sugars using a DNS-based colorimetric system. The colour produced is proportional to enzyme activity. Units are expressed as nanomoles per second of xylose reducing sugar equivalents (BXU) . 16. Samples were analyzed for neutral protease activity using casein as a substrate at pH 7.5 and 50°C. Amino acid determination is carried out on the filtrate/supernatant using Folin's reagent which serves as a phenol colour developer. The quantity of amino acid released is proportional to enzyme activity in the original sample. 17. Association of Official Analytical Chemists . 1951 . Calcium in Animal Feed method 935.13 in Official Methods of Analysis . Assoc. Anal. Chem. , Washington, DC . 18. Association of Official Analytical Chemists . 1970 . Animal Feeds. Phosphorus method 7.095-7.098 in Official Methods of Analysis . Assoc. Anal. Chem. , Washington, DC . 19. Association of Official Analytical Chemists . 2001 . Protein in Animal Feed, Forage, Grain and Oilseeds. 990.03 in Official Methods of Analysis . Assoc. Anal. Chem. , Washington, DC . 20. JMP Pro . 2016 . Version 13.0.0 ed . SAS Institute, Inc. Cary, NC . 21. Protease units are defined as the amount of protease that liberates 1 umol para-nitroaniline (pNA) from 1 mM Suc-Ala-Ala-Pro-Phe-pNa per minute at pH 9 and 37°C . 22. Yuan L. , Wang M. , Zhang X. , Wang Z. . 2017 . Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers . PLoS One . 12 : e0173941 . Google Scholar CrossRef Search ADS PubMed 23. Walk C. L. , Cowieson A. J. , Remus J. C. , Novak C. L. , McElroy A. P. . 2011 . Effects of dietary enzymes on performance and intestinal goblet cell number of broilers exposed to a live coccidia oocyst vaccine . Poult. Sci. 90 : 91 – 98 . Google Scholar CrossRef Search ADS PubMed 24. Schramm V. G. , Durau J. F. , Barrilli L. N. E. , Sorbara J. O. B. , Cowieson A. J. , Feliz A. P. , Maiorka A. . 2017 . Interaction between xylanase and phytase on the digestibility of corn and a corn/soy diet for broiler chickens . Poult. Sci. 96 : 1204 – 1211 . Google Scholar PubMed 25. Cowieson A. J. , Roos F. F. . 2014 . Bioefficacy of a mono-component protease in the diets of pigs and poultry: a meta-analysis of effect on ileal amino acid digestibility . J. Appl. Anim. Nutr. 2 : 1 – 8 . Google Scholar CrossRef Search ADS 26. Beaulac K. R. , Bedford M. R. , van Kessel A. , Schwean-Lardner K. , Kautzman M. E. , Abbott D. A. , Classen H. L. . 2015 . High levels of dietary phytase increase growth and nutrient digestibility in young broilers . Poult. Sci. 94 ( E-Suppl.1 ): 32 . 27. Masey O’Neill V. H. , Smith J. A. , Bedford M. R. . 2014 . Multicarbohydrase enzymes for non-ruminants . Asian Australas. J. Anim. Sci 27 : 290 – 301 . Google Scholar CrossRef Search ADS PubMed 28. Santos dos T. T. , Masey O’Neill H. V. , Gonzalez-Ortiz G. , Camacho-Fernandez D. , Lopez-Coello C. . 2017 . Xylanase, protease and superdosing phytase interactions in broiler performance, carcass yield and digesta transit time . Anim. Nutr. 3 : 121 – 126 . Google Scholar CrossRef Search ADS 29. Lee S. , Bedford M. R. , Walk C. L. . 2017 . Meta-analysis: explicit value of mono-component proteases in monogastric diets . Poult. Sci. http://dx.doi.org/10.3382/ps/pey042 (accessed on 18 April 2018) . © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Applied Poultry ResearchOxford University Press

Published: May 14, 2018

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