High doses of phytase on growth performance and apparent ileal amino acid digestibility of broilers fed diets with graded concentrations of digestible sulfur amino acids

High doses of phytase on growth performance and apparent ileal amino acid digestibility of broilers fed diets with graded concentrations of digestible sulfur amino acids C. L. Walk∗,1 and S. V. Rama Rao† ∗

AB Vista, Marlborough, Wiltshire SN8 4AN, UK; and † Sri Ramadhootha Poultry Research Farm Pvt Ltd, Hyderabad 500030, India increased to ≥96% in the diet. Phytase increased (P < 0.05) BWG and improved (P < 0.05) FCR, regardless of the percent dgM+C in the diet. In experiment 2, overall (hatch to day 21) BWG increased (quadratic, P < 0.05), and FCR was improved (quadratic, P < 0.05) as dgM+C increased to ≥96% in the diet. Phytase increased FI (P < 0.05) and BWG (P < 0.05) and improved FCR (P < 0.05), regardless of the percent dgM+C in the diet. In the absence of phytase, the AID of all amino acids was greatest (quadratic, P < 0.05) in birds fed between 89 and 96% dgM+C. However, in the presence of phytase the AID of all amino acids was greatest (quadratic, P < 0.05) in birds fed 82% dgM+C and greater at all levels of dgM+C than birds fed diets without phytase (dgM+C × phytase, P < 0.05). In conclusion, phytase improved AID of all amino acids and improved growth performance regardless of the level of dgM+C in the diet.

ABSTRACT Two experiments of the same design were conducted to determine the influence of phytase on performance and apparent ileal digestibility (AID) of amino acids in broilers fed graded concentrations of digestible sulfur amino acids (dgM+C). Cobb 400 male broilers were allocated to 1 of 10 diets consisting of 5 basal diets formulated at 75, 82, 89, 96, or 103% of the Cobb 400 dgM+C requirements for each feeding phase. Phytase was included in each basal diet at 0 or 2,000 FTU/kg. In experiment 1, 33 birds/pen from hatch to day 42 were fed a 2-phase feeding program with 10 replicate pens/diet. In experiment 2, there were 26 birds/pen from hatch to day 21 and 8 replicate pens/diet. Data were analyzed as a 5 × 2 factorial and means separated using orthogonal contrasts. In experiment 1, overall (hatch to day 42) feed intake (FI) decreased (linear, P < 0.05), body weight gain (BWG) increased (quadratic, P < 0.05), and feed conversion ratio (FCR) was improved (quadratic, P < 0.05) as dgM+C

Key words: apparent ileal digestibility, broiler, cysteine, methionine, phytase 2018 Poultry Science 0:1–12 http://dx.doi.org/10.3382/ps/pey218

INTRODUCTION

In poultry, methionine and cysteine have distinctly different functions in the body. Both methionine and cysteine are important for protein synthesis and subsequently meat yield. However, cysteine, is reversibly converted to cystine, and also has roles in feather and skin development and is a large structural component of mucin (Bansil and Turner, 2006). The efficiency of converting methionine to cysteine has been described as approximately 80% (Graber and Baker, 1971). Cysteine accounts for approximately 44 to 47% of the total sulfur amino acid requirement (Kalinowski et al., 2003a, 2003b). However, the efficiency of methionine use, and subsequent requirement for cysteine, can be affected by the age and rate of feathering of broilers (Kalinowski et al., 2003a, 2003b) and the dispensable amino acid nitrogen concentration in the diet (Fatufe and Rodehutscord, 2005). Other factors in the diet, such as phytate, may also impact the methionine and cysteine requirement of broilers. The anti-nutrient effect of phytate on methionine and cysteine requirements may be associated with

Methionine has various functions in the animal, with 3 main roles as an essential component for protein synthesis, as a methyl donor, and as a precursor of cysteine (Graber and Baker, 1971). Cysteine is generally considered a dispensable amino acid because the requirement for cysteine can be met through the transsulfuration of methionine. However, in protein-free diets, the nitrogen-sparing action of methionine was the result of cystine (the dimer of cysteine) formation from cysteine rather than meeting the methionine requirement (Webel and Baker, 1999). Therefore, methionine and cysteine are considered together in feed formulation and when describing nutrient requirements.  C 2018 Poultry Science Association Inc. Received February 19, 2018. Accepted April 30, 2018. 1 Corresponding author: AB Vista 3, Woodstock Court, Blenheim Road, Marlborough, Wiltshire SN8 4AN, UK; Tel.: +44 01762 517655; E-mail: [email protected]

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WALK AND RAMA RAO

reductions in endogenous protease activity by phytate and phytate esters (Yu et al., 2012), binding of phytate to proteins, peptides or amino acids as reviewed by Selle et al. (2012), and an increase in mucin secretion and endogenous amino acid losses by 14 to 55% (Cowieson and Ravindran, 2007). Phytase supplementation is reported to improve amino acid digestibility (Cowieson and Bedford, 2009) and reduce endogenous amino acid losses (Cowieson and Ravindran, 2007) through the breakdown of phytate and phytate esters, particularly at doses of phytase > 1,000 phytase units (FTU)/kg (Walk et al., 2014; Beeson et al., 2017). However, the effect of phytase on amino acid digestibility and its impact on the animals’ amino acid requirement is not the same for each amino acid. For example, the effect of phytase on methionine digestibility was 1.02% compared with that of cysteine digestibility, which was much greater at 4.60% (Cowieson and Bedford, 2009). Therefore, the objective of this set of trials was to determine the influence of superdoses of phytase on the digestible methionine and cysteine (dgM+C) requirement of broilers using growth performance, apparent ileal amino acid digestibility (AID), and digestible amino acid intake (dgAAI) as response variables.

MATERIALS AND METHODS All experimental procedures complied with Indian ethical standards for use of vertebrate animals in research.

Animals and Husbandry Experiment 1 Cobb 400 male broilers (n = 3,300) were obtained at day of hatch and placed in floor pens on clean rice husk at a stocking density of 14.4 chicks/m2 . There were 33 birds/pen and 10 replicate pens/diet. Birds were vaccinated against Newcastle disease virus and infectious bursal disease virus per label recommendations. For the entire duration of the experiment (42 d), birds were maintained on a lighting program of 23L:1D and allowed ad libitum access to feed and water. Experiment 2 Cobb 400 male broilers (n = 2,080) were obtained at day of hatch and placed in floor pens on clean rice husk at a stocking density of 14.4 chicks/m2 . There were 26 birds/pen and 8 replicate pens/diet. Birds were vaccinated against Newcastle disease virus and infectious bursal disease virus per label recommendations. For the entire duration of the experiment (21 d), birds were maintained on a lighting program of 23L:1D and allowed ad libitum access to feed and water. Dietary Treatments All diets were based on cornsoybean meal and fed in mash form (Tables 1–3). Except for Ca, P, and dgM+C, diets were formulated to meet or exceed Cobb 400 requirements (VenCobb 400

Broiler Management Guide, Cobb-Vantress Inc., Siloam Spring, AR). Dietary treatments consisted of 5 levels of dgM+C (75, 82, 89, 96, and 103% of the requirement at each feeding phase) and 2 doses of phytase (0 and 2,000 FTU/kg) arranged as a 5 × 2 factorial. Corn was exchanged with phytase where appropriate to equal 100%. The phytase was a modified and enhanced Escherichia coli 6-phytase expressed in Trichoderma reesei with an expected activity of 5,000 FTU/g (Quantum Blue, AB Vista, Marlborough, UK). One phytase unit is defined as the amount of enzyme required to release 1 μmol of inorganic P/min from sodium phytate at 37◦ C and pH 5.5.

Response Variables Experiment 1 Birds were weighed by pen prior to placement (day 0), day 21, and day 42 to determine mean BW and calculate mean BW gain (BWG). Feed addition and feed remaining were measured at day 0, feed changes (day 21), and the conclusion of the trial (day 42) to calculate feed intake (FI). Body weight gain and FI were used to calculate feed conversion ratio (FCR). Mortality was recorded daily. Any culled or dead birds were weighed. Treatment FI and thus FCR were adjusted according to the number of bird days/pen, where bird days is defined as the number of days each bird survived. Diet DM, crude protein, Ca, P, and amino acids were determined at Sciantec Analytical Services Ltd (Cawood, UK). For determination of Ca and P, diet samples were ashed at 510◦ C and then digested with a mixture of nitric and hydrochloric acid. The diluted and filtered samples were then aspirated into an ICP-OES (Perkin Elmer Optima 5300DV), and the optical emission measured at the wavelength selected for that mineral using standards of known concentration. Crude protein concentration in the diets was determined using the DUMAS method and a Leco FP528 N analyzer, and DM content was determined by gravimetry. For determination of amino acids, method 152/2009 of the European Commission (2009) was utilized and amino acids were separated by ion exchange chromatography. Phytase activity recovered in the diets was analyzed by Enzyme Services and Consultancy (Ystrad Mynach, UK) according to modified methods of Engelen et al. (2001). Experiment 2 Birds were weighed by pen prior to placement (day 0) and day 21 to determine mean BW and calculate mean BWG. Feed addition and feed remaining were measured at day 0 and the conclusion of the trial (day 21) to calculate FI. Body weight gain and FI were used to calculate FCR. Mortality was recorded daily. Any culled or dead birds were weighed. Treatment FI and thus FCR were adjusted according to the number of bird days/pen, where bird days is defined as the number of days each bird survived. On day 21, 8 birds/pen were anesthetized by exposure to CO2 gas for approximately 30 s and euthanized

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PHYTASE AND DIGESTIBLE SULFUR AMINO ACIDS Table 1. Calculated and analyzed nutrient content of the starter basal diets (experiment 1). Percent of assumed digestible M+C requirement Basal diet Ingredient, % of diet (as-fed basis) Corn Soybean meal, 48% Soybean oil Salt Limestone Dicalcium phosphate1 Sodium bicarbonate Lysine-HCl DL-methionine Threonine Premix2 Inert (corn/phytase)3 Nutrient composition, % Crude protein ME, kcal/kg Dry matter Calcium Total phosphorus Available phosphorus Total methionine Total cysteine Total methionine + cysteine Total lysine Digestible methionine Digestible cysteine Digestible methionine + cysteine Digestible lysine Sodium Chloride Analyzed nutrient composition, % Crude protein Calcium Total phosphorus Total lysine Total methionine Total cysteine Diet without phytase, FTU/kg Diet with phytase, FTU/kg

75%

82%

89%

96%

103%

61.36 34.11 1.59 0.28 0.75 1.21 0.20 0.22 0.06 0.03 0.15 0.04

61.41 34.02 1.57 0.28 0.75 1.21 0.20 0.22 0.12 0.03 0.15 0.04

61.46 33.93 1.54 0.28 0.75 1.21 0.20 0.22 0.18 0.04 0.15 0.04

61.51 33.84 1.51 0.28 0.75 1.21 0.20 0.23 0.24 0.04 0.15 0.04

61.56 33.73 1.48 0.28 0.75 1.22 0.20 0.23 0.31 0.04 0.15 0.04

22.00 3,000.00 87.35 0.78 0.62 0.30 0.38 0.38 0.77 1.37 0.35 0.33 0.68 1.25 0.18 0.26

22.00 3,000.00 87.35 0.78 0.62 0.30 0.44 0.38 0.83 1.37 0.42 0.32 0.74 1.25 0.18 0.26

22.00 3,000.00 87.36 0.78 0.62 0.30 0.50 0.38 0.89 1.37 0.48 0.32 0.80 1.25 0.18 0.26

22.00 3,000.00 87.36 0.78 0.62 0.30 0.57 0.38 0.95 1.37 0.54 0.32 0.86 1.25 0.18 0.26

22.00 3,000.00 87.36 0.78 0.62 0.30 0.64 0.38 1.02 1.37 0.61 0.32 0.93 1.25 0.18 0.26

22.95 0.78 0.55 1.46 0.42 0.38 < 50 1,670

22.70 0.81 0.58 1.45 0.50 0.38 < 50 2,030

23.50 0.77 0.56 1.45 0.54 0.40 < 50 1,840

23.65 0.74 0.54 1.47 0.61 0.40 < 50 2,030

23.80 0.83 0.58 1.50 0.69 0.40 < 50 1,740

1

Dicalcium phosphate supplied 18.5% P and 22% Ca. Supplied per kilogram of diet: iron (ferrous sulfate), 34 mg; manganese (manganese sulfate), 38 mg; zinc (zinc sulfate), 34 mg; copper (basic copper chloride), 6 mg; iodine (calcium iodate), 0.8 mg; selenium (sodium selenite), 113 μ g; vitamin A, 9.4 MIU; vitamin D3 2.1 MIU; vitamin E, 22.5 mg; vitamin B12 , 11 μ g; riboflavin, 3.8 mg; niacin, 25 mg; d-pantothenic acid, 11 mg; vitamin K, 1.5 mg; folic acid, 0.8 mg; vitamin B6 , 1.9 mg; thiamine, 1.5 mg; biotin, 60 μ g. 3 Corn was added in place of phytase in the diets without phytase supplementation. The phytase used was Quantum Blue (AB Vista, Marlborough, UK) with an expected activity of 5,000 FTU/g. 2

by cervical dislocation for ileal digesta collection. Digesta was obtained from the entire ileum (defined as Meckel’s diverticulum to the ileo-ceco-colic junction) and pooled/pen for determination of AID of amino acids. Digesta was dried at 110◦ C for 24 h and ground to pass a 1 mm screen. Dried, ground digesta and the experimental diets were analyzed for amino acids (method 982.30), crude protein (method 984.13 A-D), and chromium (method 990.08) according to AOAC (2006) at the University of Missouri Agricultural Experiment Station (Columbia, MO). Phytase activity recovered in the diets was analyzed by Enzyme Services and Consultancy (Ystrad Mynach) according to modified methods of Engelen et al. (2001).

Calculations and Statistical Analyses Apparent ileal amino acid digestibility was calculated using chromium ratios in the diets and digesta (Ravindran et al., 1999).  AA 

AID (%) =

Cr





diet − AA ileal  AA  Cr Cr diet



× 100

where ( AA Cr )diet = the ratio of amino acid to chromium in the diet; and ( AA Cr )ileal = the ratio of amino acid to chromium in the ileal digesta.

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WALK AND RAMA RAO Table 2. Calculated and analyzed nutrient content of the grower basal diets (experiment 1). Percent of assumed digestible M+C requirement Basal diet

75%

Ingredient, % of diet (as-fed basis) Corn Soybean meal Soya oil Salt Limestone Dicalcium phosphate1 Sodium bicarbonate DL-methionine Premix2 Inert (corn/phytase)3 Nutrient composition, % Crude protein ME, kcal/kg Dry matter Calcium Total phosphorus Available phosphorus Total methionine Total cysteine Total methionine + cysteine Total lysine Digestible methionine Digestible cysteine Digestible methionine + cysteine Digestible lysine Sodium Chloride Analyzed nutrient composition, % Crude protein Calcium Total phosphorus Total lysine Total methionine Total cysteine Diet without phytase, FTU/kg Diet with phytase, FTU/kg

82%

89%

96%

103%

61.50 31.52 4.54 0.29 0.80 0.92 0.20 0.02 0.15 0.04

61.54 31.45 4.52 0.29 0.80 0.92 0.20 0.07 0.15 0.04

61.58 31.37 4.49 0.29 0.80 0.92 0.20 0.13 0.15 0.04

61.63 31.29 4.47 0.29 0.80 0.92 0.20 0.19 0.15 0.04

61.66 31.22 4.45 0.29 0.80 0.93 0.20 0.24 0.15 0.04

20.50 3,200.00 87.63 0.72 0.55 0.25 0.33 0.36 0.69 1.12 0.30 0.31 0.61 1.01 0.18 0.22

20.50 3,200.00 87.63 0.72 0.55 0.25 0.38 0.36 0.74 1.12 0.35 0.31 0.66 1.01 0.18 0.22

20.50 3,200.00 87.64 0.72 0.55 0.25 0.44 0.36 0.80 1.12 0.41 0.31 0.72 1.00 0.18 0.22

20.50 3,200.00 87.64 0.72 0.55 0.25 0.50 0.36 0.86 1.12 0.47 0.31 0.78 1.00 0.18 0.22

20.50 3,200.00 87.65 0.72 0.55 0.25 0.55 0.36 0.91 1.12 0.52 0.31 0.83 1.00 0.18 0.22

22.15 0.67 0.50 1.14 0.36 0.37 < 50 1,880

21.35 0.65 0.49 1.12 0.40 0.37 < 50 1,970

21.95 0.71 0.46 1.16 0.48 0.38 < 50 1,860

21.90 0.70 0.49 1.12 0.53 0.36 < 50 1,840

23.25 0.67 0.49 1.15 0.57 0.37 < 50 1,820

1

Dicalcium phosphate supplied 18.5% P and 22% Ca. Supplied per kilogram of diet: iron (ferrous sulfate), 51 mg; manganese (manganese sulfate), 57 mg; zinc (zinc sulfate), 51 mg; copper (basic copper chloride), 9 mg; iodine (calcium iodate), 1.1 mg; selenium (sodium selenite), 171 μ g; vitamin A, 14 MIU; vitamin D3 3.2 MIU; vitamin E, 34 mg; vitamin B12 , 17 μ g; riboflavin, 5.7 mg; niacin, 38 mg; pantothenic acid, 17 mg; vitamin K, 2.3 mg; folic acid, 1.1 mg; vitamin B6 , 2.9 mg; thiamine, 2.3 mg; biotin, 91 μ g. 3 Corn was added in place of phytase in the diets without phytase supplementation. The phytase used was Quantum Blue (AB Vista, Marlborough, UK) with an expected activity of 5,000 FTU/g. 2

To calculate dgAAI in g/d, the following equation was used (Walk et al., 2018): 

dgAAI (g/day) =

(AA diet, %) ×



AID AA, % 100



100 × daily intake, g

where AA diet = the analyzed amino acid concentration of the diet and AID AA = the previously calculated apparent ileal amino acid digestibility. In both experiments, data were analyzed as a completely randomized 2 × 5 factorial design using the fit model platform of JMP 13.0 (SAS Institute, Cary, NC). Outliers were determined as 3 times the root mean square error plus or minus the mean of response. Plotting the growth performance, AID, or dgAAI data

using a normal quantile plot indicated the means were normally distributed. Pen served as the experimental unit for all parameters measured. The statistical model included dietary phytase, dgM+C, and the interaction. When differences were significant, means were separated using linear and quadratic orthogonal contrast statements. Significance was accepted at P ≤ 0.05. The dgM+C requirements were determined using the BWG, FCR, AID of Met, Cys, and M+C, and dgAAI of Met, Cys, and M+C. Dietary concentrations of dgM+C and dgM+C × dgM+C were considered continuous, independent variables to predict a linear or quadratic response of dgM+C concentration on the response variable. The final model equation was:

y = a + bx + cx2 ± d

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PHYTASE AND DIGESTIBLE SULFUR AMINO ACIDS Table 3. Calculated and analyzed nutrient content of the starter basal diets (experiment 2). Percent of assumed digestible M+C requirement Basal diet Ingredient, % of diet (as-fed basis) Corn Soybean meal, 48% Soybean oil Salt Limestone Dicalcium phosphate1 Sodium bicarbonate Lysine-HCl DL-methionine Threonine Premix2 Chromium Inert (corn/phytase)3 Nutrient composition, % Crude protein ME, kcal/kg Dry matter Calcium Total phosphorus Available phosphorus Total methionine Total cysteine Total methionine + cysteine Total lysine Digestible methionine Digestible cysteine Digestible methionine + cysteine Digestible lysine Sodium Chloride Analyzed nutrient composition, % Crude protein Total lysine Total methionine Total cysteine Diet without phytase, FTU/kg Diet with phytase, FTU/kg

75%

82%

89%

96%

103%

61.33 34.11 1.59 0.28 0.75 1.21 0.20 0.22 0.06 0.03 0.15 0.03 0.04

61.38 34.02 1.57 0.28 0.75 1.21 0.20 0.22 0.12 0.03 0.15 0.03 0.04

61.43 33.93 1.54 0.28 0.75 1.21 0.20 0.22 0.18 0.04 0.15 0.03 0.04

61.48 33.84 1.51 0.28 0.75 1.21 0.20 0.23 0.24 0.04 0.15 0.03 0.04

61.53 33.73 1.48 0.28 0.75 1.22 0.20 0.23 0.31 0.04 0.15 0.03 0.04

22.00 3,000.00 87.35 0.78 0.62 0.30 0.38 0.38 0.77 1.37 0.35 0.33 0.68 1.25 0.18 0.26

22.00 3,000.00 87.35 0.78 0.62 0.30 0.44 0.38 0.83 1.37 0.42 0.32 0.74 1.25 0.18 0.26

22.00 3,000.00 87.36 0.78 0.62 0.30 0.50 0.38 0.89 1.37 0.48 0.32 0.80 1.25 0.18 0.26

22.00 3,000.00 87.36 0.78 0.62 0.30 0.57 0.38 0.95 1.37 0.54 0.32 0.86 1.25 0.18 0.26

22.00 3,000.00 87.36 0.78 0.62 0.30 0.64 0.38 1.02 1.37 0.61 0.32 0.93 1.25 0.18 0.26

22.70 1.41 0.38 0.36 < 50 1,850

21.34 1.30 0.42 0.35 < 50 2,550

22.58 1.39 0.49 0.35 < 50 2,350

22.35 1.38 0.55 0.35 < 50 2,360

23.32 1.41 0.60 0.35 < 50 2,230

1

Dicalcium phosphate supplied 18.5% P and 22% Ca. Supplied per kilogram of diet: iron (ferrous sulfate), 34 mg; manganese (manganese sulfate), 38 mg; zinc (zinc sulfate), 34 mg; copper (basic copper chloride), 6 mg; iodine (calcium iodate), 0.8 mg; selenium (sodium selenite), 113 μ g; vitamin A, 9.4 MIU; vitamin D3 2.1 MIU; vitamin E, 22.5 mg; vitamin B12 , 11 μ g; riboflavin, 3.8 mg; niacin, 25 mg; d-pantothenic acid, 11 mg; vitamin K, 1.5 mg; folic acid, 0.8 mg; vitamin B6 , 1.9 mg; thiamine, 1.5 mg; biotin, 60 μ g. 3 Corn was added in place of phytase in the diets without phytase supplementation. The phytase used was Quantum Blue (AB Vista, Marlborough, UK) with an expected activity of 5,000 FTU/g. 2

where y = response variable, a = intercept, b = linear coefficient, c = quadratic coefficient, x = dgM+C concentration in the experimental diet, and d = without or with phytase. Phytase supplementation was considered a nominal, independent variable to predict the effect of phytase on the dgM+C requirement at each response variable. If the effect of phytase was significant, the predicted dgM+C requirement without or with phytase was determined using 50 or 95% confidence limits.

RESULTS Nutrients analyzed and phytase activity recovered in the diets were similar to formulated values (Tables 1–3).

Animal Performance and Carcass Evaluation Experiment 1 Overall mortality was 1.75% and not significantly affected by dgM+C, phytase, or the interaction (data not shown). From hatch to day 21, increasing dgM+C in the diet to 96% of the assumed requirement improved BWG (quadratic, P < 0.05) and FCR (quadratic, P < 0.05) with no effect on FI (Table 4). Phytase supplementation increased FI (P < 0.05) and BWG (P < 0.05) and improved FCR (P < 0.05; Table 4). Overall (hatch to day 42), increasing the concentration of dgM+C in the diet decreased FI (linear, P < 0.05) and improved BWG (quadratic, P < 0.05) and FCR (quadratic, P < 0.05) when fed

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WALK AND RAMA RAO

Table 4. Influence of the percent dietary digestible methionine + cysteine and superdoses of phytase on broiler performance1 from hatch to 21 d post-hatch (experiment 1). % of digestible M+C requirement in the diet (% digestible Met)2

Phytase

75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61) 75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61)

– – – – – + + + + +

SEM 75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61) SEM – + SEM dgM+C Linear Quadratic Phytase dgM+C × phytase

Feed intake, g

BW gain, g

FCR, g:g3

1,180.4 1,196.3 1,211.7 1,188.0 1,191.9 1,204.5 1,253.8 1,235.0 1,246.0 1,233.3 12.5

826.8 863.0 884.2 881.9 871.5 869.5 928.8 927.4 949.2 946.5 10.6

1.428 1.387 1.370 1.348 1.369 1.387 1.351 1.333 1.314 1.303 0.012

1,192.4 1,225.0 1,223.3 1,217.0 1,212.6 8.9

848.1 895.9 905.8 915.5 909.0 7.5

1.408 1.369 1.352 1.331 1.336 0.008

1,193.6 1,234.5 5.6

865.5 924.2 4.7

1.380 1.337 0.005

0.0783 0.2535 0.0201 < 0.0001 0.4566

< 0.0001 < 0.0001 0.0002 < 0.0001 0.4183

< 0.0001 < 0.0001 0.0076 < 0.0001 0.6701

1

Means represent the average response of 10 replicate pens/treatment and 33 birds/pen. 2 Digestible cysteine was calculated in the diets at 0.38% and therefore the dietary sulfur amino acid content increased with the inclusion of DLmethionine. 3 Mortality corrected feed: gain.

up to 96% of the assumed requirement (Table 5). Phytase supplementation increased BWG (P < 0.05) and improved FCR (P < 0.05) from 1.740 to 1.696 (Table 5). There was no effect of dgM+C × phytase on growth performance from hatch to day 21 or day 42 post-hatch. Experiment 2 Overall mortality was 1.92% and not significantly affected by dgM+C, phytase, or the interaction (data not shown). From hatch to day 21, there was no effect of dietary dgM+C on FI (Table 6). However, increasing dgM+C in the diet up to 103% of the assumed requirement improved BWG (quadratic, P < 0.05) and FCR (quadratic, P < 0.05; Table 6). Phytase supplementation increased FI (P < 0.05) and BWG (P < 0.05) and improved FCR (P < 0.05) from 1.378 to 1.317 (Table 6). There was no effect of dgM+C × phytase on growth performance from hatch to day 21.

Apparent Ileal Digestibility and Digestible Intake Apparent ileal amino acid digestibility and dgAAI were only evaluated in experiment 2 and those results

Table 5. Influence of the percent dietary digestible methionine + cysteine and superdoses of phytase on broiler performance1 from hatch to 42 d post-hatch (experiment 1). % of digestible M+C requirement in the diet

Phytase

75 82 89 96 103 75 82 89 96 103

– – – – – + + + + +

SEM 75 82 89 96 103 SEM – + SEM dgM+C Linear Quadratic Phytase dgM+C × phytase

Feed intake, g

BW gain, g

FCR, g:g2

4,220.3 4,234.9 4,184.6 4,167.3 4,152.6 4,251.0 4,277.4 4,225.3 4,180.7 4,153.8 27.1

2,316.3 2,391.3 2,412.6 2,463.5 2,459.4 2,385.8 2,501.2 2,501.1 2,513.2 2,542.4 16.2

1.822 1.771 1.726 1.692 1.688 1.782 1.710 1.690 1.664 1.634 0.007

4,235.6 4,256.1 4,205.0 4,174.0 4,153.2 19.2

2,351.1 2,446.3 2,456.9 2,488.4 2,500.9 11.5

1.802 1.741 1.708 1.678 1.661 0.005

4,191.9 4,217.6 12.1

2,408.6 2,488.7 7.3

1.740 1.696 0.003

0.0013 0.0001 0.3867 0.1378 0.9281

< 0.0001 < 0.0001 0.0011 < 0.0001 0.4390

< 0.0001 < 0.0001 < 0.0001 < 0.0001 0.1494

1 Means represent the average response of 10 replicate pens/treatment with 33 birds/pen from hatch to day 42. 2 Mortality corrected feed: gain.

are presented in Tables 7–10. Apparent ileal digestibility of methionine, cysteine, M+C, or total amino acids was influenced by dgM+C × phytase (P < 0.05; Table 7). In the absence of phytase, the AID of methionine, cysteine, M+C, and total amino acids increased (quadratic, P < 0.05) with increasing concentrations of dgM+C in the diet up to ≥ 96% of the assumed requirement. In the presence of phytase, the AID of cysteine, M+C, and total amino acids increased (quadratic, P < 0.05) with increasing concentrations of dgM+C in the diet, but the asymptote was reached at 82% of the assumed dgM+C requirement. Similarly, the AID of methionine increased (quadratic, P < 0.05) as dgM+C concentration in the diet increased to 96% of the assumed dgM+C requirement. In all cases, the AID was greater in the presence of phytase than in the absence. In the absence of phytase, the dgAAI of methionine, cysteine, and M+C increased (linear, P < 0.05) with increasing dgM+C concentration to 103% of the assumed requirement (Table 8). However, in the presence of phytase, the dgAAI of methionine, cysteine, and M+C increased (quadratic, P < 0.05) with increasing dgM+C concentration to ≥ 96, 82, or 96%, respectively, of the assumed requirement and this resulted in significant dgM+C × phytase interactions. The dgAAI of total amino acids increased (quadratic, P < 0.05) as the dgM+C concentration in the diet increased to 89%

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7

PHYTASE AND DIGESTIBLE SULFUR AMINO ACIDS Table 6. Influence of the percent dietary digestible methionine + cysteine and superdoses of phytase on broiler performance1 from hatch to 21 d post-hatch (experiment 2). % of digestible M+C requirement in the diet (% digestible Met)2

Phytase

75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61) 75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61)

– – – – – + + + + +

SEM 75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61) SEM – + SEM dgM+C Linear Quadratic Phytase dgM+C × phytase

Feed intake, g

BW gain, g

FCR, g:g3

913.9 926.6 938.4 936.6 938.6 928.8 957.1 958.7 966.9 965.9 12.8

629.1 667.7 689.6 695.0 702.6 660.8 720.9 745.2 752.6 756.0 11.8

1.453 1.388 1.361 1.349 1.337 1.408 1.329 1.287 1.285 1.279 0.01

921.3 941.8 948.6 951.7 952.3 9.1

645.0 694.3 717.4 723.8 729.3 8.3

1.430 1.359 1.324 1.317 1.308 0.008

930.8 955.5 5.7 0.1000 0.0145 0.2029 0.0033 0.9655

676.8 727.1 5.3 < 0.0001 < 0.0001 0.0013 < 0.0001 0.8036

of the assumed requirement. Phytase supplementation increased the dgAAI of total amino acids (P < 0.05) from 8.11 to 8.84 g/d. In the absence of phytase, the AID of dispensable amino acids (quadratic for all, P < 0.05) or indispensable amino acids (quadratic for all, P < 0.05) increased as the dgM+C concentration in the diet increased to 96% of the assumed requirement, except for the AID of arginine or tryptophan which was optimized at 89% of the assumed dgM+C requirement. However, in the presence of phytase the optimum AID of dispensable amino acids (quadratic for all, P < 0.05) or indispensable amino acids (quadratic for all, P < 0.05) was achieved at 82% of the assumed dgM+C requirement, except the AID of tryptophan which was optimized at 96% of the assumed dgM+C requirement. This resulted in a dgM+C × phytase (P < 0.05) on the AID of all measured dispensable amino acids (Table 9) or indispensable amino acids (Table 10).

Regression Estimates

1.378 1.317 0.005 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.8087

1 Means represent the average response of 8 replicate pens/treatment and 26 birds/pen. 2 Digestible cysteine was calculated in the diets at 0.38% and therefore the dietary sulfur amino acid content increased with the inclusion of DLmethionine. 3 Mortality corrected feed: gain.

Regression equations indicated that there was a quadratic (P < 0.05) effect of dgM+C concentration in the diet on BWG (experiments 1 and 2), FCR (experiments 1 and 2), and the AID of methionine, cysteine, and M+C, and dgAAI of cysteine and M+C (experiment 2; Table 11). The effect of dgM+C concentration in the diet was linear (P < 0.05) for dgAAI of Met (experiment 2). Phytase supplementation influenced (P < 0.05) the dgM+C requirements for BWG (experiments 1 and 2), FCR (experiments 1 and 2), and AID of methionine, cysteine, and M+C (experiment 2) and

Table 7. Influence of the percent dietary digestible methionine + cysteine and superdoses of phytase on apparent ileal methionine, cysteine, methionine + cysteine, and total amino acid digestibility1 of 21-day-old broilers (experiment 2). % of digestible M+C requirement in the diet (% digestible Met)2

Phytase

Met, %

Cys, %

M + C, %

Total AA, %

75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61) 75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61)

– – – – – + + + + +

86.12 90.72 92.21 93.58 93.92 92.08 94.53 94.66 94.71 94.57 0.37 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0003

64.99 67.57 69.49 71.06 68.43 76.13 81.00 77.81 73.87 73.73 0.94 0.0004 < 0.0001 < 0.0001 0.0008 0.0030 0.0001 0.0030

75.85 80.36 82.67 84.57 84.63 84.32 88.30 87.61 86.72 86.81 0.56 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0575 0.0003

80.89 83.28 84.42 85.16 83.49 87.28 89.91 88.37 86.81 86.71 0.49 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0077 0.0038

SEM dgM+C Phytase dgM+C × phytase Linear dgM+C 0 FTU Quadratic dgM+C 0 FTU Linear dgM+C 2000 FTU Quadratic dgM+C 2000 FTU 1

Means represent the average response of 8 replicate pens/treatment and 8 birds/pen. Digestible cysteine was calculated in the diets at 0.38% and therefore the dietary sulfur amino acid content increased with the inclusion of DL-methionine. 2

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8

WALK AND RAMA RAO Table 8. Influence of the percent dietary digestible methionine + cysteine and superdoses of phytase on apparent digestible methionine, cysteine, methionine + cysteine, and total amino acid intake (g/d) of 21-day-old broilers (experiment 2). % of digestible M+C requirement in the diet (% digestible Met)2

Phytase

dgMet intake, g/d

dgCys intake, g/d

dgM + C intake, g/d

dgAA intake, g/d

75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61) 75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61)

– – – – – + + + + +

0.139 0.168 0.194 0.213 0.256 0.155 0.177 0.216 0.253 0.257 0.003

0.099 0.101 0.106 0.108 0.107 0.121 0.129 0.128 0.123 0.119 0.002

0.238 0.269 0.299 0.321 0.363 0.276 0.306 0.344 0.375 0.375 0.005

7.85 8.01 8.18 8.21 8.28 8.61 8.63 9.21 9.06 8.68 0.13

0.147 0.172 0.205 0.234 0.256 0.002

0.110 0.115 0.117 0.115 0.113 0.002

0.257 0.288 0.322 0.348 0.369 0.004

8.23 8.32 8.70 8.64 8.48 0.09

0.194 0.211 0.001

0.104 0.124 0.001

0.298 0.335 0.002

8.11 8.84 0.06

< 0.0001 < 0.0001 0.2819 < 0.0001 < 0.0001 < 0.0001 0.0674 < 0.0001 0.0011

0.0284 0.2728 0.0024 < 0.0001 0.0026 0.0017 0.3141 0.0919 0.0010

< 0.0001 < 0.0001 0.0520 < 0.0001 0.0015 < 0.0001 0.4849 < 0.0001 0.0008

0.0020 0.0064 0.0085 < 0.0001 0.1774

SEM 75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61) SEM – + SEM dgM+C Linear Quadratic Phytase dgM+C × phytase Linear dgM+C 0 FTU Quadratic dgM+C 0 FTU Linear dgM+C 2000 FTU Quadratic dgM+C 2000 FTU 1

Means represent the average response of 8 replicate pens/treatment and 8 birds/pen. Digestible cysteine was calculated in the diets at 0.38% and therefore the dietary sulfur amino acid content increased with the inclusion of DL-methionine. 2

dgAAI of methionine, cysteine, and M+C (experiment 2; Table 11). In the absence of phytase, the predicted dgM+C requirement for BWG from hatch to day 21 (experiment 1 or 2) or hatch to day 42 (experiment 1) was 95.3, 98.3, or 100.3%, respectively (Table 12). Phytase supplementation reduced the predicted dgM+C requirement for BWG from hatch to day 21 (experiments 1 and 2) or hatch to day 42 (experiment 1) to 76.1, 80.1, or 81.1%, respectively. The predicted dgM+C requirement to optimize FCR from hatch to day 21 (experiments 1 and 2) or hatch to day 42 (experiment 1) was 99.5, 98.3, or >103.0%, respectively, in the absence of phytase and reduced to 80.8, 81.8, or 88.8%, respectively, in the presence of phytase. The predicted dgM+C requirement to optimize AID and dgAAI of Met was 97.7 and >103.0%, respectively, in the absence of phytase and 80.6 and 98.7%, respectively, in the presence of phytase. The predicted dgM+C requirement to optimize AID and dgAAI of Cys was 88.5 and 90.6%, respectively, in the absence of phytase and <75.0% in the presence of phytase. Fi-

nally, using the AID and dgAAI of M+C, the predicted M+C requirement in the absence of phytase was 96.1 or >103.0%, respectively. However, in the presence of phytase, the predicted dgM+C requirement was 76.1 or 91.9% based on the AID or the dgAAI of M+C, respectively. Thereby for all parameters evaluated, less dgM+C was required in the diet to achieve optimum BWG, FCR, and AID of amino acids in the presence of phytase.

DISCUSSION Nutrient and Enzyme Recoveries The objective of the current trial was to determine the influence of phytase on the dgM+C requirement of broilers. In this regard, phytase recoveries in the experimental diets were within expected ranges, when assay variation and product overages are considered. Total amino acids, protein, Ca, and P analyzed in the diets are reflective of formulated values and

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PHYTASE AND DIGESTIBLE SULFUR AMINO ACIDS

Table 9. Influence of the percent dietary digestible methionine + cysteine and superdoses of phytase on apparent ileal dispensable amino acid digestibility1 of 21-day-old broilers (experiment 2). % of digestible M+C requirement in the diet (% digestible Met)2

Phytase

75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61) 75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61)

– – – – – + + + + +

SEM dgM+C Phytase dgM+C × phytase Linear dgM+C 0 FTU Quadratic dgM+C 0 FTU Linear dgM+C 2000 FTU Quadratic dgM+C 2000 FTU

Asp, %

Ser, %

Glu, %

Pro, %

Gly, %

Ala, %

Tyr, %

80.04 82.13 83.27 84.27 82.10 87.02 89.70 87.86 86.42 85.87 0.54

78.94 81.65 82.74 82.75 80.39 85.02 88.44 86.45 84.34 84.02 0.54

86.30 87.76 88.36 89.07 87.48 91.03 93.01 91.83 90.95 91.02 0.43

77.72 79.21 80.40 81.39 79.18 84.09 87.70 85.50 83.11 84.25 0.62

74.52 77.65 79.35 80.19 78.00 82.68 86.29 83.86 81.44 81.90 0.59

79.04 81.56 82.94 83.74 81.72 85.83 88.99 87.10 85.28 85.53 0.60

80.85 84.48 85.38 86.60 84.39 88.21 89.98 89.26 87.11 86.80 0.50

< 0.0001 < 0.0001 < 0.0001 0.0005 0.0001 0.0018 0.0040

< 0.0001 < 0.0001 < 0.0001 0.0213 < 0.0001 0.0006 0.0003

0.0008 < 0.0001 0.0024 0.0087 0.0004 0.1352 0.0326

0.0009 < 0.0001 < 0.0001 0.0111 0.0016 0.0321 0.0297

< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0009 0.0055

< 0.0001 < 0.0001 < 0.0001 0.0002 0.0001 0.0256 0.0124

< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0006 0.0038

1

Means represent the average response of 8 replicate pens/treatment and 8 birds/pen. Digestible cysteine was calculated in the diets at 0.38% and therefore the dietary sulfur amino acid content increased with the inclusion of DL-methionine. 2

Table 10. Influence of the percent dietary digestible methionine + cysteine and superdoses of phytase on apparent ileal indispensable amino acid digestibility1 of 21-day-old broilers (experiment 2). % of digestible M+C requirement in the diet (% digestible Met)2

Phytase

75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61) 75 (0.35) 82 (0.42) 89 (0.48) 96 (0.54) 103 (0.61)

– – – – – + + + + +

SEM dgM+C Phytase dgM+C × phytase Linear dgM+C 0 FTU Quadratic dgM+C 0 FTU Linear dgM+C 2000 FTU Quadratic dgM+C 2000 FTU

Thr, %

Val, %

Iso, %

Leu, %

Phe, %

Lys, %

His, %

Arg, %

Trp, %

72.66 75.56 77.76 78.66 76.76 81.12 84.98 82.63 80.28 79.99 0.61

75.48 78.82 80.01 80.75 78.40 83.32 86.72 84.62 82.17 82.54 0.58

79.93 82.52 83.77 84.79 82.73 87.11 89.97 88.05 87.04 86.88 0.53

81.90 83.94 84.89 85.90 84.16 88.00 90.61 89.23 87.84 87.91 0.54

82.47 84.52 85.46 86.44 84.84 88.77 91.19 89.81 88.57 88.19 0.48

87.14 89.77 91.38 91.48 90.91 92.35 93.81 93.20 92.84 91.78 0.44

83.27 86.62 86.75 87.06 86.71 89.21 91.04 90.50 88.33 87.28 0.42

88.72 91.48 92.09 92.04 91.00 93.99 94.78 93.42 92.72 91.84 0.42

88.67 91.47 91.51 90.75 91.43 92.46 92.57 92.73 90.24 89.13 0.41

< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0005 0.0005

< 0.0001 < 0.0001 < 0.0001 0.0001 < 0.0001 0.0015 0.0046

< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0460 0.0116

0.0002 < 0.0001 0.0002 0.0003 0.0004 0.0873 0.0136

< 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0001 0.0154 0.0034

< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.1341 0.0051

< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

< 0.0001 < 0.0001 < 0.0001 0.0002 < 0.0001 < 0.0001 0.0921

< 0.0001 0.0125 < 0.0001 0.0004 0.0015 < 0.0001 0.0014

1

Means represent the average response of 8 replicate pens/treatment and 8 birds/pen. Digestible cysteine was calculated in the diets at 0.38% and therefore the dietary sulfur amino acid content increased with the inclusion of DL-methionine. 2

indicate that the expected reductions in nutrients were achieved.

Effect of Digestible Sulfur Amino Acids Feeding diets marginal in Ca and P, without or with phytase, and increasing the dgM+C concentration in

the diet from 75 to 103% of the assumed requirement quadratically increased BWG and improved FCR. The cysteine concentration in the diets (formulated and analyzed) did not change as the dgM+C concentration in the diet increased from 75 to 103%. Therefore, the responses reported herein are predominantly associated with increasing the concentration of supplemental DL-methionine in the diet. However, dietary

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WALK AND RAMA RAO

Table 11. Regression equations and coefficients of the influence of digestible methionine + cysteine requirements in broilers fed superdoses of phytase from hatch to 42 d post-hatch (experiments 1 and 2). Trait (Y)

Regression equation1

R2

± phytase

dgM+C P-value

P-value

Linear

Quadratic

Phytase

Experiment 1 Digestible methionine + cysteine requirements (X) for broilers from hatch to 21 d post-hatch BW gain, g Y = –524.4 + 30.2 × X – 0.158 × X2 0.57 0.49 FCR, g:g Y = 2.552–0.024 × X + 0.000123 × X2

29.4 0.022

< 0.0001 0.0025

0.0002 0.0066

< 0.0001 < 0.0001

Digestible methionine + cysteine requirements (X) for broilers from hatch to 42 d post-hatch 0.62 BW gain, g Y = 367.4 + 42.4 × X – 0.210 × X2 FCR, g:g Y = 3.202–0.029 × X + 0.000134 × X2 0.85

40.1 0.022

0.0003 < 0.0001

0.0013 < 0.0001

< 0.0001 < 0.0001

Experiment 2 Digestible methionine + cysteine requirements (X) for broilers from hatch to 21 d post-hatch BW gain, g Y = –739.7 + 29.9 × X – 0.152 × X2 0.61 0.73 FCR, g:g Y = 3.448–0.044 × X + 0.0002 × X2 AID methionine, % Y = 6.93 + 1.79 × X – 0.009 × X2 0.73 0.64 AID cysteine, % Y = –49.47 + 2.78 × X – 0.0157 × X2 AID M+C, % Y = –30.46 + 2.42 × X – 0.013 × X2 0.72 0.93 dgAAI methionine, g/d Y = –0.254 + 0.006 × X – 0.00001 × X2 dgAAI cysteine, g/d Y = –0.0995 + 0.0048 × X – 0.0000026 × X2 0.71 0.90 dgAAI M+C, g/d Y = –0.347 + 0.0109 × X – 0.0000038 × X2

25.2 0.030 1.33 4.10 2.57 0.009 0.010 0.019

0.0003 < 0.0001 < 0.0001 0.0008 < 0.0001 0.0215 0.0041 0.0050

0.0010 < 0.0001 < 0.0001 0.0007 < 0.0001 0.3926 0.0046 0.0738

< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

1 Regression equations determined only if there was a significant linear or quadratic effect of dgM+C as determined by feeding broilers diets containing 75 to 103% of the assumed dgM+C requirement.

Table 12. Predicted response of increasing the digestible methionine + cysteine concentration in the diets of broilers fed diets without or with superdoses of phytase from hatch to 42 d post-hatch1 (experiments 1 and 2). Predicted response, % dgM+C requirement, % Trait (Y)

Mean

– phytase

+ phytase

Predicted response using 95% confidence limits, %

dgM+C replaced with phytase, %

dgM+C requirement, % – phytase

+ phytase

dgM+C replaced with phytase, %

Experiment 1 Hatch to 21 d post-hatch BW gain, g FCR, g:g

887.4 1.355

95.3 99.5

76.1 80.8

19.2 18.7

94.7 ≥103.0

80.3 88.9

14.4 > 14.1

Hatch to 42 d post-hatch BW gain, g FCR, g:g

2457.6 1.684

100.6 ≥103.0

81.1 88.8

19.5 > 14.2

≥103.0 ≥103.0

87.1 93.1

> 15.9 > 9.9

18.2 16.5 17.1 > 13.5 20.0 > 4.3 > 15.6 > 11.1

≥103.0 99.9 98.3 88.8 95.7 ≥103.0 90.6 ≥103.0

85.8 85.9 82.3 < 75.0 79.4 101.0 < 75.0 95.8

> 17.2 14.0 16.0 > 13.8 16.3 > 2.0 > 15.6 > 7.2

Experiment 2 Hatch to 21 d post-hatch BW gain, g FCR, g:g AID methionine, % AID cysteine, % AID M+C, % dgAAI methionine, g/d dgAAI cysteine, g/d dgAAI M+C, g/d 1

704.9 1.337 93.0 69.9 83.5 0.25 0.11 0.35

98.3 98.3 97.7 88.5 96.1 ≥103.0 90.6 ≥103.0

80.1 81.8 80.6 < 75.0 76.1 98.7 < 75.0 91.9

Values only determined if there was a significant effect of phytase from Table 11.

methionine is converted to cysteine and cysteine corresponds to almost half of the total sulfur amino acid requirement (Kalinowski et al., 2003a, 2003b); therefore, the results can be described as the determination of the dgM+C requirement. Evaluation of the performance results indicate the graded concentrations of dgM+C in the diets were sufficient to elicit a dose response, allow for predictions of the dgM+C requirement for broilers from hatch to day 21 and day 42, and allow for the determination of the effect of phytase on the dgM+C requirement.

The lack of a significant effect of dgM+C on FI from hatch to day 21 has been previously reported (Kalinowski et al., 2003a) and could indicate a different nutrient, such as P, was more limiting than dgM+C in the starter diets. Others have reported a significant increase in FI as the concentration of methionine or cystine increased in the diet (Graber and Baker, 1971; Fatufe and Rodehutscord, 2005). Gous (2007) stated in nutrientdeprived diets that birds will eat to meet the requirement at the limit of “fill” which will subsequently influence BWG and FCR. The linear decreased in FI from

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PHYTASE AND DIGESTIBLE SULFUR AMINO ACIDS

hatch to day 42 as the dgM+C concentration in the diet increased suggests dgM+C were the most limiting nutrients in the diets and the birds increased intake in an effort to meet their dgM+C requirements. This is confirmed with the significant and quadratic effects of the concentration of dgM+C in the diet on BWG and FCR for all phases. From hatch to day 21, the optimum BWG and FCR was achieved at 95 to 99% of the assumed dgM+C requirement and this was even higher from hatch to day 42 at 100 to > 103% of the assumed dgM+C requirement. This corresponds to a dgM+C concentration in the diet of 0.86 to 0.90% in the starter phase and > 0.80% in the grower phase, depending on the parameter evaluated. These estimated requirements are higher but within the range of the dgM+C requirements reported for male Cobb broiler chicks, which decreased from 0.87 to 0.66% in the pre-starter to finisher phases (de Castro Goulart et al., 2011) but similar to the total methionine and cysteine requirements of male Ross broilers at 0.94 or 0.89% 3 wk post-hatch or 0.88 to 0.83% at 6 wk post-hatch, depending on fast- or slow feathering, respectively (Kalinowski et al., 2003a, 2003b). Feathering requires a large proportion of cysteine, and previous authors have reported no effect of slow- or fast feathering on the cysteine requirements of 3-wk-old broilers (Kalinowski et al., 2003a). However, the same authors reported fast-feathering broilers had a higher requirement for cysteine than slow-feathering broilers from 3 to 6-wk post-hatch (Kalinowski et al., 2003b). In the current trial, the increase in the assumed dgM+C requirement from < 99% at day 21 to > 103% at day 42 indicates a shift in the birds’ nutrient partitioning or an additional nutrient requirement which increased the birds’ dgM+C need to enable the bird to continue with to protein synthesis (i.e., growth) and now also promote feather production, which could account for up to 10% of the total protein requirement (Stillborn et al., 1994).

Effect of Phytase Superdosing dietary phytase is a relatively new term and could be described as feeding > 1,500 FTU/kg of a highly efficacious microbial phytase. Previous authors have reported significant improvements in FCR of broilers fed diets marginally deficient in P and Ca and fed superdoses of phytase (Walk et al., 2013, 2014; Gautier et al., 2018). The benefits in feed efficiency have been attributed to near complete destruction of dietary phytate in the gizzard (Walk et al., 2014) and significant improvements in the AID of amino acids (Beaulac et al., 2015) which resulted in improvements in BWG without significant increases in FI. In the current trials, the diets were marginally deficient in both Ca and P. This was done to mimic typical dietary conditions of phytase supplementation and cannot be ignored when discussing the effects of phytase on the estimated dgM+C requirements. Feed

11

intake was decreased in birds fed diets not supplemented with phytase, but only in the starter phase and this could indicate that dietary P was the most limiting nutrient in the starter diets, an effect that was lost in the grower phase. However, when considering the significant quadratic effects of increasing dgM+C in the diets, at all phases and in both experiments, it can be concluded the growth responses in this experiment are associated with differences in the dgM+C concentrations in the diets, rather than solely a P and phytase response. Phytase supplementation significantly increased BWG and the AID of all amino acids measured, improved FCR, and reduced the estimated dietary concentration of dgM+C needed to optimize BWG, FCR, and the AID of methionine, cysteine, and M+C. Previous authors have reported significant increases in AID of amino acids as phytase supplementation in the diet increased (Sommerfeld et al., 2018), with the effect of phytase on the digestibility of dispensable amino acids being greater than the effect of phytase on the digestibility of indispensable amino acids (Beaulac et al., 2015). In the current experiments, from hatch to day 21, phytase could spare between 17 and 19% of the dgM+C concentration in the diet and from hatch to day 42, phytase could spare > 14% of the dgM+C concentration in the diets. This effect of phytase is quite substantial and cannot be described through reductions in mucin and endogenous losses alone. However, through nearly complete destruction of phytate, phytase would reduce endogenous amino acid losses while also improving endogenous enzyme efficiency, nutrient digestion, and efficiency of nutrient utilization for protein synthesis and feather production. Within the gastrointestinal tract, the methionine and cysteine concentration of ileal endogenous protein is approximately 3.3%, with the methionine and cysteine content of endogenous secretions such as mucin, endogenous enzymes, and bile accounting for 10.8, 2.8 and 2.4%, respectively, of the total ileal endogenous protein (Ravindran, 2016). Assuming up to 94% phytate destruction by the ileum with superdoses of phytase (Sommerfeld et al., 2018), the effect of phytase on sparing endogenous amino acid losses could account for approximately 3% of the response reported in this paper. This effect may have also accounted for some of the significant improvements in AID of both methionine (+1.3%) and cysteine (+4.1%). It could be estimated that 4 to 7% of the effect of phytase reported in this paper was associated with the sparing of dgM+C losses and improvements in AID through phytate destruction. The remaining effects of phytase on sparing of dgM+C may be associated with an improvement in the overall efficiency of utilization of dgM+C and the other amino acids in the diet. For example, phytase supplementation had a large and significant effect on the AID of both indispensable (+4.2% for tryptophan to +12.5% for threonine) and dispensable amino acids (+6.0% for glutamate to +11.1% for glycine), and

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WALK AND RAMA RAO

this was greatest at lower (≤ 82%) concentrations of dgM+C in the diet. The efficiency of methionine use is significantly impacted by the dispensable amino acid nitrogen content of the diet, whereby the maximum efficiency of methionine retention was 85% for birds fed a low protein diet and 78% for birds fed a normal protein diet (Fatufe and Rodehutscord, 2005). This means the methionine concentrations required to achieve a plateau in growth or efficiency were higher for birds fed the normal protein diet compared with birds fed the low protein diet supplemented with dispensable amino acids (Fatufe and Rodehutscord, 2005). Therefore, in the current set of experiments, the improvement in the AID of dispensable amino acids with superdoses of phytase may have increased the efficiency of methionine use. This would have resulted in a plateau in growth or efficiency at a lower dietary dgM+C concentration than required for birds fed diets without phytase. The increase in both dispensable and indispensable amino acid digestibility from phytase supplementation, the reduced concentrations of dietary methionine, and the model employed (Fatufe and Rodehutscord, 2005) will all impact the estimated dgM+C requirements. In the current experiments, the results indicate growth performance and AID of amino acids was achieved at 89 to > 103% of the dgM+C concentration in the diet, in the absence of phytase. However, supplementation of the diet with superdoses of phytase significantly increased growth performance, improved efficiency, and amino acid digestibility, particularly in diets formulated to contain between 75 and 82% of the assumed dgM+C requirement. The effect of superdoses of phytase on the dgM+C sparing in the diet was between 14 and 19% for growth performance and AID of amino acids.

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