Animals and Management

The Animal Experimentation Committee of the Scotland’s Rural College approved all bird handling and sample collection procedures.

A total of 336 male broiler chicks (Ross 308) were brooded together and fed a nutrient adequate pre-experimental starter diet (Table 3-1) from d 0 to 14 in experiment 1 and 2 or from d 0 to 24 in experiment 3. On d 14, birds were weighed individually and divided into 3 groups of similar bodyweight consisting of 126, 126 or 84 birds for experiment 1, 2 or 3, respectively. In each experiment, birds were allocated to one of the experimental diets in a randomized complete block design using d 14 bodyweight as blocking criterion and transferred to metabolism cages on d 14. Each treatment had seven replicate cages and three birds per replicate cage. Birds were weighed individually on d 14 and at the end of the experimental period (d21 or 28). In experiment 2 and 3, birds were euthanized by cervical dislocation on d 28 to allow collection of ileal digesta samples. Birds were provided ad libitum access to the experimental diets and water throughout the pre- and experimental periods. The birds were reared in a house with facilities to control temperature, light, and humidity. Room temperature was maintained at 35^oC, 32^oC, 27^oC and 23^oC for day 1 to 7, 8 to 14, 15 to 21 and 22 to 28, respectively. Titanium dioxide (TiO2) was added to the diets (3 g/kg of diet) as an indigestible marker to enable determination of ME content and P and AA utilisation by the index method.

84 3.2.2 Dietary Treatments and Sample Collection

Experiment 1

The chemical composition of the wheat-DDGS used in the current study is presented in Table 3-2. The energy value of wheat-DDGS for broilers was determined using a total of six diets in experiment 1. Wheat-DDGS was incorporated in a wheat-soybean meal diet at 3 levels (0, 300, or 600 g/kg) without- or with added XAP (0 or 0.25 g/kg) to make six diets in total. At a rate of 0.25 g/kg, the XAP (Danisco Animal Nutrition, Marlborough, UK) supplied 2000, 200 and 4000 U of xylanase, amylase and protease, respectively per kg of diet. The xylanase is a Endo-1,4-beta-xylanase produced by a Trichoderma longibrachiatum and expressed in the same organism. The amylase was produced by Bacillus amyloliquifaciens and expressed in Bacillus subtilis. The subtilisin (protease) was derived from Bacillus subtilis. These 3 enzymes were produced separately and later blended to produce the xylanase-amylase-protease (XAP) admixture. One unit (U) of xylanase was defined as the quantity of the enzyme that liberates one mmol of xylose equivalent per minute. One unit of amylase was defined as the amount of the enzyme catalysing the hydrolysis of one mmol glucosidic linkage per minute and one protease unit was defined as the quantity of the enzyme that solubilised one mg of azo-casein per minute.

Because maintaining a similar ratio of energy-yielding components is important when using the regression method, energy-yielding ingredients such as wheat, soybean meal (SBM), gluten meal and soy oil were substituted with wheat-DDGS in a way that their ratios were the same across all the experimental diets. These ratios were 1.93, 11.1, 10.4, 5.37, 5.76, and 1.07 for wheat:SBM, wheat:gluten meal, wheat:soyoil, SBM:soyoil, SBM:gluten meal, and soyoil:gluten meal, respectively for the experimental diets presented in Table 3-3.

Experimental diets were fed from d 15 to 21. Excreta was collected daily from each cage for three days (d 18 to 20), dried and pooled for each cage for the analysis of gross energy (GE), dry matter (DM), N and Ti to determine AME and AMEn.

85 Table 3-1. Ingredient and nutrient composition of pre-experimental standard

diet.

2Vitamin/mineral premix supply per kilogram of diet: vitamin A, 16,000 IU; vitamin D3, 3,000 IU;

vitamin E, 25 IU; vitamin B1, 3 mg; vitamin B2, 10 mg; vitamin B6, 3 mg; vitamin B12, 15 µg; hetra, 5 mg; nicotinic acid, 60 mg; pantothenic acid, 14.7 mg; folic acid, 1.5 mg; Biotin, 125 µg; choline chloride, 25 mg; iron, 20 mg; copper, 10 mg; manganese, 100 mg; cobalt, 1.0 mg; zinc, 82.222 mg; iodine, 1 mg;

selenium, 0.2 mg; and molybdenum, 0.5 mg.

86 Table 3-2. Analysed nutrient composition of wheat Distillers’ Dried Grains with

Solubles (as-is basis)

87 Experiment 2

A total of six diets were used in experiment 2. Three levels of wheat-DDGS (200, 400 or 600 g/kg) was incorporated in a corn-starch based diet (wheat-DDGS being the only source of P) without- or with added phytase. The phytase (Danisco Animal Nutrition, Marlborough, UK) was added to the diet to supply 1000 FTU/kg. The phytase was derived from Escherichia coli and expressed in Schizosaccharomyces pombe. One phytase unit was defined as the quantity of enzyme required to liberate 1 µmol of inorganic P per minute, at pH 5.5 from an excess of 15 µM sodium phytate at 37^oC. The ingredient and analysed chemical compositions of the experimental diets are presented in Table 3-4. Diets were fed from d 15 to 21. Excreta samples were collected daily for 3 days (d 18 to 20) for the determination of total tract P utilisation. On d 21, all birds were euthanized by cervical dislocation and ileal digesta samples were collected from the Meckel’s diverticulum to approximately 1 cm proximal to the ileo-cecal junction by flushing with distilled water. Ileal digesta samples were pooled per cage and stored frozen (-20^oC) pending chemical analysis.

88 Table 3-3. Ingredient and analysed nutrient composition of experimental diets to determine metabolisable energy value of wheat-DDGS for broilers with- or without added xylanase, amylase and protease.

Level of dietary wheat distillers dried grains with solubles, g/kg

Without XAP With added XAP

Item 0 300 600 0 300 600

Ingredients, g/kg

Wheat, White 561 385.2 209.2 561 385.2 209.2

Soybean meal -48% 291.2 199.9 108.6 291.2 199.9 108.6

Soybean oil 54.2 37.2 20.2 54.2 37.2 20.2

Gluten meal 38.6 22.7 7.0 31.6 15.7 0

DDGS 0 300 600 0 300 600

XAP premix¹ 0 0 0 7.0 7.0 7.0

Others² 55.0 55.0 55.0 55.0 55.0 55.0

Analysed energy and nutrient composition³

Dry matter, g/kg 880 875 870 880 880 870

Gross energy, MJ/kg 17.4 17.6 17.8 17.3 17.8 17.8

CP (N x 6.25), g/kg 223 252 275 226 256 276

Xylanase activity, U/kg - - - 1423 1399 1442

Amylase activity, U/kg - - - 262 262 262

Protease activity, U/kg <100 <100 <100 3064 3064 3064

1XAP premix made with gluten meal as carrier; formulated to supply 2000U/kg of xylanase, 200U/kg of amylase and 4000U/kg of protease.

2Others consists of 18.5 g/kg of Limestone (38% Ca); 14 g/kg of Dicalcium phosphate (Contain 21.3% Ca and 18.7% P); 1 g/kg of Common salt; 3 g/kg of Vitamin/mineral premix (vitamin A, 16,000 IU; vitamin D3, 3,000 IU; vitamin E, 25 IU; vitamin B1, 3 mg;

vitamin B2, 10 mg; vitamin B6, 3 mg; vitamin B12, 15 µg; hetra, 5 mg; nicotinic acid, 60 mg; pantothenic acid, 14.7 mg; folic acid, 1.5 mg; Biotin, 125 µg; choline chloride, 25 mg; iron, 20 mg; copper, 10 mg; manganese, 100 mg; cobalt, 1.0 mg; zinc, 82.222 mg;

iodine, 1 mg; selenium, 0.2 mg; and molybdenum, 0.5 mg); 1 g/kg of DL-Metionine; 2.5 g/kg of L-Lysine HCl; 15 g/kg of marker premix (prepared as 1 g of titanium dioxide added to 4 g of gluten meal).

3Values are means of duplicate analyses.

89 Table 3-4. Ingredient and chemical composition of experimental diets to determine phosphorus utilisation of wheat-DDGS for

broilers.

Inclusion level of dietary wheat distillers’ dried grains with solubles, g/kg

Without Phytase With added Phytase

Item 200 400 600 200 400 600

Ingredients, g/kg

Corn starch 526 303.5 87 516 293.5 77

DDGS 200 400 600 200 400 600

Soybean oil 18 36 48 18 36 48

Limestone 4.5 9 13.5 4.5 9 13.5

Others¹ 251.5 251.5 251.5 251.5 251.5 251.5

Phytase premix² 0 0 0 10 10 10

Analysed composition³, g/kg

Dry matter 880 890 880 880 890 885

Phosphorus 2.3 3.3 4.0 2.0 2.9 4.2

Calcium 3.6 5.4 6.3 3.5 4.7 6.9

Copper 0.011 0.012 0.013 0.010 0.010 0.015

Iron 0.512 0.129 0.138 0.087 0.117 0.147

Magnesium 0.8 1.2 1.4 0.7 1.1 1.6

Sodium 3.3 4.2 4.6 3.2 3.8 5.0

Phytase activity, FTU/kg <50 <50 <50 962 767 822

1Others consist of: 100 g/kg of dextrose; 130 g/kg of sucrose; 2.5 g/kg of vitamin/mineral premix (supply per kilogram of diet: vitamin A, 16,000 IU;

vitamin D3, 3,000 IU; vitamin E, 25 IU; vitamin B1, 3 mg; vitamin B2, 10 mg; vitamin B6, 3 mg; vitamin B12, 15 µg; hetra, 5 mg; nicotinic acid, 60 mg; pantothenic acid, 14.7 mg; folic acid, 1.5 mg; Biotin, 125 µg; choline chloride, 25 mg; iron, 20 mg; copper, 10 mg; manganese, 100 mg; cobalt, 1.0 mg; zinc, 82.2 mg; iodine, 1 mg; selenium, 0.2 mg; and molybdenum, 0.5 mg); 4 g/kg of common salt; 15 g/kg of marker premix (prepared as 1 g Titanium dioxide added to 4 g of gluten meal).

2Phytase premix made with cornstarch as carrier; Formulated to supply 1000 FTU/kg. ³Values are means of duplicate analyses

90 (Danisco Animal Nutrition, Marlborough, UK). One protease unit was defined as the quantity of the enzyme that solubilises one mg of azo-casein per minute. Basal ileal endogenous AA flow from birds fed a NFD without- or with protease was used to correct AIAAD values to SIAAD. The ingredient compositions of the experimental diets are presented in Table 3-5.

The analysed CP and AA compositions for the diets are presented in Table 3-6. The assay diets were formulated to contain 22% CP and balanced for mineral and vitamins to meet breeder nutrient specifications. Experimental diets were fed from d 25 to 28. Because of health and welfare issues associated with feeding birds NFD, experimental diets were fed for three days in experiment 3. A 3-d feeding period is optimal to fulfil the objectives of the experiment (Kluth and Rudehurscord, 2010). On d 28, ileal digesta samples were collected from the Meckel’s diverticulum to approximately 1 cm proximal to the ileo-cecal junction by flushing with distilled water. Ileal digesta samples were pooled for each cage and stored frozen (-20 ^oC) prior to chemical analysis.