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Phytase in starter and grower diets of White Pekin ducks
Phytase in starter and grower diets of White Pekin ducks O. Adeola1 Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 Ducks had free access to diets and water during the 42day study. Feeding the low-P NC diet to ducks reduced (P < 0.01) gain and feed intake compared with the PC diet in both starter and grower phases. Supplementing the NC diet with phytase resulted in both linear and quadratic improvements (P < 0.05) in gain, feed intake, and G: F. Feeding the low-P NC diet to ducks reduced (P < 0.01) tibia ash compared with the PC diet. There were both linear and quadratic increases (P < 0.05) in tibia ash with phytase supplementation. Supplementing the NC diet with phytase resulted in both linear and quadratic increases (P < 0.001) in ileal digestibility and retention of P in both the starter and grower phases. The results of this study showed that phytase was efficacious in hydrolyzing phytate P for bone mineralization and growth of ducks through the starter and grower periods.
ABSTRACT The growth performance and phosphorus utilization responses of ducks to phytase were investigated during the starter and grower phases. Fivehundred-seventy-six one-day-old drakes with an average initial BW of 55 g were grouped by BW into 8 blocks of 6 pens and assigned to 48 pens with 12 ducks per pen. The 6 dietary treatments consisted of: 1) positive control (PC), adequate in all nutrients with 4.5 g non-phytate phosphorus (nPP)/kg starter diet or 3.5 g nPP/kg grower diet; 2) negative control (NC), adequate in all other nutrients except phosphorus with 3.0 g nPP/kg starter diet or 2.0 g nPP/kg grower diet; 3) the NC plus phytase at 500 units/kg diet; 4) the NC plus phytase at 1,000 units/kg diet; 5) the NC plus phytase at 1,500 units/kg diet; and 6) the NC plus phytase at 15,000 units/kg diet. Starter and grower diets were fed from d 1 to 15 and d 15 to 43 post hatching, respectively.
Key words: bone ash, ducks, growth performance, phosphorus, phytase 2018 Poultry Science 0:1–7 http://dx.doi.org/10.3382/ps/pex352
INTRODUCTION
Ducks differ in P digestion from other avian species (Rodehutscord and Dieckmann, 2005), and a paucity of performance data exists for ducks fed phosphorusmarginal diets supplemented with phytase for an entire 42-day growth period. The objectives of the study reported here were to evaluate the growth performance, bone mineralization, as well as phosphorus utilization responses of White Pekin ducks when fed phosphorusmarginal diets supplemented with phytase for 42 days.
The phosphorus in cereal grains and oilseeds is bound in phytin, the collective term for mixed salts of magnesium, calcium, and potassium of phytic acid, the main storage form of plant phosphorus. Phytin constitutes up to 3% of many of the oilseeds and cereals used in animal feeds (Reddy et al., 1982). If hydrolyzed, phytin in feed can be a good source of phosphorus to the animal. Because intestinal phytase produced by non-ruminants is inadequate, in vivo hydrolysis of phytate is minimal, and therefore utilization of phosphorus in cereal grains and oilseed meals is limited. Consequently, much of the phytate-bound phosphorus in the diet is passed out in animal waste (Adeola, 1999). Furthermore, phytic acid has strong chelating potential in the gut and complexes Ca and Zn, thus reducing their bioavailability (Nolan et al., 1987). Adding microbial phytase to diets has been demonstrated to increase phytate hydrolysis and the availability of phosphorus in broiler chickens (Dilger et al., 2004) and ducks (Orban et al., 1999; Rodehutscord et al., 2006; Adeola, 2010).
MATERIALS AND METHODS Ducks and Diets All protocols used in the study were approved by the Purdue University Animal Care and Use Committee. Seven-hundred-sixty-eight day-old male ducks with an average initial BW of 55 g were wing-banded, weighed, and assigned to 48 pens with 16 ducks per pen. The ducks were weighed individually, sorted in decreasing order of body weight, and assigned to 6 diets such that the average initial weight of ducks was similar across dietary treatments with 16 ducks per pen and 8 pens per diet. Diets were randomly assigned to pens in each block of 6 pens. Ingredient and calculated composition of diets are presented in Table 1. The dietary treatments
C 2017 Poultry Science Association Inc. Received April 2, 2017. Accepted October 30, 2017. 1 Corresponding author: [email protected]
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ADEOLA
Table 1. Ingredient composition of the starter (d 1 to 15) and grower (d 15 to 43) experimental diets. Starter diets
Item, g/kg Corn Soybean meal Soy oil Salt Limestone Dicalcium phosphate Vitamin-mineral premix1 DL-Methionine L-Lysine.HCl Chromic oxide premix2 Phytase premix3 Phytase premix4
Positive control (PC)
Grower diets
Negative NC + NC + NC + NC + control 500 1,000 1,500 15,000 (NC) units/kg units/kg units/kg units/kg
Positive control (PC)
Negative NC + NC + NC + NC + control 500 1,000 1,500 15,000 (NC) units/kg units/kg units/kg units/kg
557.8 360.0 30.0 4.0 9.4 17.5 3.0
565.4 360.0 30.0 4.0 9.8 9.5 3.0
560.4 360.0 30.0 4.0 9.8 9.5 3.0
555.4 360.0 30.0 4.0 9.8 9.5 3.0
550.4 360.0 30.0 4.0 9.8 9.5 3.0
550.4 360.0 30.0 4.0 9.8 9.5 3.0
721.1 205.0 25.0 4.0 10.0 13.5 3.0
729.1 205.0 25.0 4.0 10.5 5.0 3.0
724.1 205.0 25.0 4.0 10.5 5.0 3.0
719.1 205.0 25.0 4.0 10.5 5.0 3.0
714.1 205.0 25.0 4.0 10.5 5.0 3.0
714.1 205.0 25.0 4.0 10.5 5.0 3.0
3.3 0.0 15.0
3.3 0.0 15.0
3.3 0.0 15.0
3.3 0.0 15.0
3.3 0.0 15.0
3.3 0.0 15.0
1.9 1.5 15.0
1.9 1.5 15.0
1.9 1.5 15.0
1.9 1.5 15.0
1.9 1.5 15.0
1.9 1.5 15.0
0.0 0.0
0.0 0.0
5.0 0.0
10.0 0.0
15.0 0.0
0.0 15.0
0.0 0.0
0.0 0.0
5.0 0.0
10.0 0.0
15.0 0.0
0.0 15.0
Total 1,000 Calculated nutrients and energy Crude protein, g/kg 220 ME, kcal/kg 3048 Ca, g/kg 8.0 Total P, g/kg 7.1 nPP, g/kg 4.5 Ca:P 1.13
1,000
1,000
1,000
1,000
1,000
1000
1,000
1,000
1,000
1,000
1,000
220 3074 6.5 5.6 3.0 1.16
220 3074 6.5 5.6 3.0 1.16
220 3074 6.5 5.6 3.0 1.16
220 3074 6.5 5.6 3.0 1.16
220 3074 6.5 5.6 3.0 1.16
160 3175 7.0 5.8 3.5 1.20
161 3202 5.5 4.3 2.0 1.28
161 3202 5.5 4.3 2.0 1.28
161 3202 5.5 4.3 2.0 1.28
161 3202 5.5 4.3 2.0 1.28
161 3202 5.5 4.3 2.0 1.28
1 Supplied the following per kg of diet: vitamin A, 5484 IU; vitamin D3 , 2643 ICU; vitamin E,11 IU; menadione sodium bisulfite, 4.38 mg; riboflavin, 5.49 mg; d-pantothenic acid, 11 mg; niacin, 44.1 mg; choline chloride, 771 mg; vitamin B12 , 13.2 μ g; biotin, 55.2 μ g; thiamine mononitrate, 2.2 mg; folic acid, 990 μ g; pyridoxine hydrochloride, 3.3 mg; I, 1.11 mg; Mn, 66.06 mg; Cu, 4.44 mg; Fe, 44.1 mg; Zn, 44.1 mg; Se, 300 μ g. 2 Prepared as 1 g chromic oxide added to 4 g of corn. 3 Phytase premix prepared by serial dilution with corn to contain 100 phytase units/g. 4 Phytase premix prepared by serial dilution with corn to contain 1,000 phytase units/g.
consisted of: 1) positive control (PC), adequate in all nutrients with 8.0 g Ca and 4.5 g non-phytate phosphorus (nPP)/kg starter diet or 7.0 g Ca and 3.5 g nPP/kg grower diet; 2) negative control (NC), adequate in all other nutrients except Ca and P with 6.5 g Ca and 3.0 g nPP/kg starter diet or 5.5 g Ca and 2.0 g nPP/kg grower diet; 3) the NC plus phytase at 500 units/kg diet; 4) the NC plus phytase at 1,000 units/kg diet; 5) the NC plus phytase at 1,500 units/kg diet; 6) the NC plus phytase at 15,000 units/kg diet. Ducks had free access to mash diets. One unit of phytase activity is defined as the quantity of enzyme required to hydrolyze 1 μmol of inorganic P/min, at pH 5.5, from an excess of 1.5 mM sodium phytate at 37◦ C (International Union of Biochemistry, 1979).
Management Flock of ducks was examined daily for any variation in appearance or behavior for 42 days. Weight gain and feed intake were monitored weekly. Each pen was equipped with one hanging cylindrical feeder, one flat plastic feed tray, one satellite waterer and a multinozzle drip nipple waterer. On d 5, the satellite water was removed from pens after ducks had been trained to drink from drip nipples; the plastic feed tray was removed at this time. A daily lighting regimen of 23 h light and 1 h dark was maintained. Room temperatures from d 1 to 8, d 8 to 15, d 15 to 22, d 22 to
29, d 29 to 35, and d 35 to 43 were kept at 35◦ C, 31◦ C, 29◦ C, 27◦ C, 24◦ C, and 22◦ C, respectively. Temperature in the room was controlled by ventilation fans. Ileal digesta and excreta were collected as previously described (Adeola et al., 1997; Adeola, 2010). Four ducks from each pen were fitted with excreta collection bags from d 15 to 18 or d 43 to 46. The collection bags were attached to the retainer rings that were sutured to the vents. The excreta collected were used to determine apparent total tract nutrient retention. On d 18 or 43, the ducks fitted with collection bags were euthanatized by carbon dioxide and intestinal digesta were collected from the Meckel’s diverticulum to the ileo-cecal junction by flushing with distilled water. Ileal digesta and excreta were stored at −20◦ C immediately after collection. Ileal digesta and excreta from ducks within a pen were pooled for the analysis of Cr, P, N, Ca, and energy. Tibia were excised from each duck and stored at −20◦ C.
Chemical and Statistical Analyses Samples for analyses were processed and analyzed as previously described by Adeola (2010). Diets, freezedried ileal digesta, and excreta were ground to pass through a 1 mm screen and dried at 105◦ C in a drying oven (Precision Scientific Co., Chicago, IL) for 24 h for dry matter determination. Gross energy was determined in a bomb calorimeter (Parr 1261 bomb
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PHYTASE IN DUCK DIETS Table 2. Analyzed composition of the experimental diets. Diets
Dry matter, %
Crude protein, %
Gross energy, kcal/g
Phytase units/kg
Ca, %
P, %
87.47 87.89 87.62 88.72 87.84 87.98
24.16 23.56 24 23.97 23.91 23.89
4.081 4.113 4.121 4.104 4.038 4.035
171 80 697 1272 1646 14,968
0.97 0.81 0.79 0.80 0.81 0.80
0.78 0.61 0.59 0.61 0.61 0.60
87.55 87.51 87.96 87.27 87.67 87.86
15.69 15.56 15.84 16.19 15.88 15.92
4.041 4.079 4.141 4.06 3.974 4.019
186 93 455 1404 1319 14,331
0.81 0.65 0.63 0.65 0.65 0.64
0.56 0.42 0.43 0.41 0.42 0.40
Starter diets Positive control (PC) Negative control (NC) NC + 500 phytase units/kg NC + 1,000 phytase units/kg NC + 1,500 phytase units/kg NC + 15,000 phytase units/kg Grower diets Positive control (PC) Negative control (NC) NC + 500 phytase units/kg NC + 1,000 phytase units/kg NC + 1,500 phytase units/kg NC + 15,000 phytase units/kg
calorimeter, Parr Instruments Co., Moline, IL) using benzoic acid as a calibration standard. The combustion method (Leco model FP-2000 N analyzer, Leco Corp., St. Joseph, MI) using EDTA as a calibration standard was employed in N analysis. Samples were digested [nitric/perchloric wet ash, AOAC 935.13 A (a)] (AOAC International, 2000) and Cr concentration was determined (Spectronic 21D, Milton Roy Company, Rochester, NY) using the method of Fenton and Fenton (1979). Total inorganic P (AOAC Official Method 985.01(A, B, D) concentration was determined by colorimetric analysis (Packard SpectraCountTM , Model # AS1000, Meriden, CT) and Ca (AOAC Official Method 985.01(A, B, D) concentration was by atomic absorption spectrophotometry (AOAC International, 2000). Phytase activity was determined by the method of Engelen et al. (1994). Tibia were defatted in a soxhlet extractor for 6 h, dried to a constant weight at 100◦ C, weighed, and then ashed in a muffle furnace at 600◦ C for 16 hours. Apparent ileal digestibility or utilization coefficient for nutrients and energy were calculated as follows: C = 1 − [(Cd/Co)
∗
(No/Nd)]
Where Cd is the concentration of Cr in the diet; Co is the concentration of Cr in the excreta or ileal digesta output; No is the concentration of nutrient or energy in the excreta or ileal digesta output; and Nd is the concentration of nutrient or energy in the diet. The product of C and the gross energy (kcal/g) concentration of the diet gives the metabolizable energy (kcal/g) or ileal digestible energy (kcal/g) of the diet. The data were analyzed using the General Linear Model procedure of SAS (SAS, 2006) in a randomized complete block design with pen as the experimental unit. Contrasts of PC vs. NC diets, and linear and quadratic contrasts were used to examine responses to supplemental graded levels of phytase at 0, 500, 1000, 1500, and 15,000 units/kg. Contrast coefficients used for unequally spaced levels were generated using the interactive matrix language procedure of SAS (2006), and statistical significance was determined at an α level of 0.05.
RESULTS The analyzed chemical composition and phytase activity of the 10 diets used in both starter and grower phases of the current study are presented in Table 2. The phytase-supplemented starter diets were formulated to contain 500, 1,000, 1500 or 15,000 units/kg analyzed at 697, 1,272, 1,646, or 14,968 units/kg, respectively. Corresponding values for the grower diets were 455, 1,404, 1,319, or 14,331 units/kg. Analyzed CP, Ca, and P were close to formulated values (Table 2). Feeding the low-phosphorus NC diet to ducks reduced (P < 0.01) gain, feed intake, and G: F compared with the PC diet, regardless of whether it was d 1 to 15, 15 to 29, 29 to 43, or 1 to 43 except for G: F during d 1 to 15 (Table 3). Supplementing the low-phosphorus NC diet with phytase resulted in both linear and quadratic improvements (P < 0.05) in gain, feed intake, and G: F. Feeding the low-P NC diet to ducks reduced (P < 0.01) tibia ash compared with the PC diet. There were both linear and quadratic increases (P < 0.05) in tibia ash with phytase supplementation (Table 3). There was no difference between the PC and NC diet in ileal digestibility except for Ca in which ileal digestibility was greater (P < 0.001) in the NC than the PC diet during the starter phase (Table 4). There were quadratic decreases (P < 0.05) in both ileal digestible energy and ileal N digestibility as phytase supplementation of the NC diet increased to 15,000 units/kg diet. Adding phytase to the NC diet linearly and quadratically increased (P < 0.001) the ileal digestibility of phosphorus. Total tract phosphorus retention and ME were lower (P < 0.05) in the NC than in the PC diet, but greater for Ca retention in NC than in the PC diet (Table 4). There was a quadratic decrease (P < 0.05) in Ca retention as phytase addition to the NC diet increased to 15,000 units/kg diet. Supplementing the lowphosphorus NC diet with phytase resulted in both linear and quadratic increases (P < 0.001) in phosphorus retention. The ileal digestibility and total tract utilization of components of the diets fed during the grower phase are presented in Table 5. Digestibility of DM, energy, N,
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ADEOLA
Table 3. Growth performance and tibia ash content of ducks fed positive control (PC), negative control (NC), and NC supplemented with phytase.1 P-value2 Phytase supplementation, Units/kg of diet Items
PC
Initial weight, g Days 1 to 15 Gain, g Feed intake, g G:F, g/kg Days 15 to 29 Gain, g Feed intake, g G:F, g/kg Days 29 to 43 Gain, g Feed intake, g G:F, g/kg Days 1 to 43 Gain, g Feed intake, g G:F, g/kg Tibia ash, %
NC
NC + 1,000
NC + 500
NC + 1,500
Phytase NC + 15,000
SEM
PC vs. NC
L
Q
1.00
1.00
0.99
55
55
55
55
55
55
0.08
551 706 781
485 584 831
527 645 816
527 662 797
544 690 788
577 702 823
11 13 13
< 0.01 < 0.01 0.01
< 0.01 < 0.01 0.40
< 0.01 < 0.01 0.01
1099 2351 467
763 1754 435
960 2106 455
997 2187 456
1045 2289 457
1089 2287 476
19 35 6
< 0.01 < 0.01 < 0.01
< 0.01 < 0.01 < 0.01
< 0.01 < 0.01 0.03
1330 4725 282
855 3887 224
1133 4130 275
1227 4515 272
1279 4682 274
1394 4606 304
34 130 9
< 0.01 < 0.01 < 0.01
< 0.01 0.02 < 0.01
< 0.01 < 0.01 < 0.01
2974 7750 384 47.5
2083 6009 348 42.5
2620 6881 381 44.3
2747 7344 374 46.7
2858 7613 376 46.9
3060 7595 403 48.8
44 137 7 1.404
< 0.01 < 0.01 < 0.01 0.02
< 0.01 < 0.01 < 0.01 0.02
< 0.01 < 0.01 0.04 0.03
1
Data are means of 8 replicate pens per diet. Probability value of the contrast of positive control vs. negative control diet (PC vs. NC), linear contrast (L), and quadratic contrast (Q). For L and Q, coefficients for unequally spaced levels were generated using Proc IML of SAS (2006). 2
Table 4. Ileal digestibility and total tract retention of ducks fed positive control (PC), negative control (NC), and NC supplemented with phytase on day 18.1 P-value2 Phytase supplementation, Units/kg of diet Items
Phytase
PC
NC
NC + 500
NC + 1,000
NC + 1,500
NC + 15,000
SEM
PC vs. NC
L
Q
69.2 70.2 3.20 76.9 44.6 42.5
69.3 69.8 3.20 78.0 40.6 57.3
70.8 71.7 3.36 78.4 48.9 59.5
67.8 67.9 3.11 75.8 47.8 59.5
68.8 68.4 3.07 75.7 54.1 60.0
68.1 68.0 3.06 75.4 65.4 54.7
1.3 1.3 0.060 1.0 2.0 3.3
0.95 0.81 0.98 0.44 0.17 < 0.001
0.40 0.28 0.04 0.11 < 0.001 0.26
0.46 0.24 0.04 0.05 < 0.001 0.52
Total tract retention DM, % 75.3 Energy, % 78.1 ME, kcal/g 3.51 N, % 65.8 P, % 44.3 Ca, % 49.4
73.7 75.1 3.29 66.4 39.4 69.0
74.8 77.1 3.500 66.9 48.6 63.3
72.4 74.1 3.29 71.6 47.3 54.3
73.0 74.7 3.34 67.1 49.4 53.0
72.4 74.9 3.36 65.7 51.8 57.9
1.7 1.5 0.065 4.3 1.5 4.8
0.50 0.16 0.02 0.92 0.03 0.01
0.55 0.77 0.99 0.66 < 0.001 0.56
0.61 0.53 0.89 0.68 < 0.001 0.01
Ileal digestibility DM, % Energy, % DE, kcal/g N, % P, % Ca, %
1
Data are means of 8 replicate cages per diet. Probability value of the contrast of positive control vs. negative control diet (PC vs. NC), linear contrast (L), and quadratic contrast (Q). For L and Q, coefficients for unequally spaced levels were generated using Proc IML of SAS (2006). 2
and P at the end of the ileum, as well as ileal digestible energy were not different between the PC and NC diets but ileal Ca digestibility was greater (P < 0.001) in the NC than in the PC diet. There were both linear and quadratic reductions (P < 0.05) in ileal digestible energy as well as a linear decrease (P = 0.05) in ileal N digestibility in response to supplementing the NC diet with 500, 1,000, 1500, or 15,000 phytase units/kg. The response (P < 0.001) in ileal phosphorus digestibility to phytase was strong, and both linear and quadratic (Table 5). There was no difference in the utilization of DM, energy, or N between ducks fed the PC or NC diets. However, ducks fed the PC diet retained more
phosphorus (P < 0.05) but less Ca (P < 0.05) than those fed the low-phosphorus NC diet. Quadratic reduction (P = 0.01) in Ca utilization with phytase supplementation of the low-phosphorus NC diet was observed. As in ileal phosphorus digestibility, there were both linear and quadratic increases (P < 0.001) in phosphorus utilization with phytase addition to the low-phosphorus NC diet (Table 5).
DISCUSSION Phytase supplementation of the diets of nonruminant animals improves the utilization of phytate
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PHYTASE IN DUCK DIETS
Table 5. Ileal digestibility and total tract retention of ducks fed positive control (PC), negative control (NC), and NC supplemented with phytase on day 46.1 P-value2 Phytase supplementation, FTU/kg of diet Items
Phytase
PC
NC
NC + 500
NC + 1,000
NC + 1,500
NC + 15,000
SEM
PC vs. NC
L
Q
70.6 71.7 3.27 78.3 45.5 43.4
70.7 71.2 3.26 79.6 40.4 58.4
72.2 73.1 3.43 80.1 49.9 60.7
69.3 69.3 3.18 77.6 50.3 60.7
70.4 70.0 3.14 77.6 56.4 61.4
69.5 69.5 3.13 77.4 66.8 55.9
1.33 1.32 0.061 1.02 2.04 3.35
0.97 0.83 1.00 0.45 0.15 < 0.001
0.41 0.29 0.04 0.11 < 0.001 0.27
0.47 0.24 0.04 0.05 < 0.001 0.53
Total tract retention DM, % 77.2 Energy, % 79.8 ME, kcal/g 3.61 N, % 67.6 P, % 45.2 Ca, % 50.4
75.2 76.6 3.42 67.7 38.7 70.4
76.5 78.0 3.51 68.4 49.6 64.7
74.0 75.7 3.43 73.0 48.7 55.4
74.7 76.9 3.41 68.8 50.9 54.2
73.9 76.5 3.44 66.8 52.9 59.3
1.74 1.52 0.066 4.39 1.53 4.92
0.56 0.79 1.01 0.67 < 0.001 0.57
0.62 0.54 0.91 0.69 < 0.001 0.01
Ileal digestibility DM, % Energy, % DE, kcal/g N, % P, % Ca, %
0.51 0.16 0.02 0.94 0.03 < .0001
1
Data are means of 8 replicate cages per diet. Probability value of the contrast of positive control vs. negative control diet (PC vs. NC), linear contrast (L), and quadratic contrast (Q). For L and Q, coefficients for unequally spaced levels were generated using Proc IML of SAS (2006). 2
phosphorus and ultimately reduces the amount of phosphorus flowing into the environment from animal waste. Results of studies abound in the literature on phytase supplementation of the diets of broiler chickens from day-old to market weight. However the same cannot be said of ducks for which results of only a few studies have been published but for various stages of the life cycle, such as parts of the starter period or portions of the grower period, and not for the entire life cycle from day-old to market weight. In the current experiment, growth performance responses of ducks for a 42-day life cycle and nutrient utilization responses to phytase supplementation were investigated. In each of the 3 2-week periods of d 1 to 15, d 15 to 29, and d 29 to 43, as well as the overall period of d 1 to 43, ducks responded to the phosphorus-adequate diets with improvements in weight gain, feed intake, and feed efficiency when compared with the low-phosphorus diets. This is a clear indication that phosphorus was indeed a limiting nutrient for the growth performance of ducks used in the current study. This observation is consistent with previous reports of low-phosphorusinduced reduction in growth performance of ducks and broiler chickens (Orban et al., 1999; Dilger et al., 2004; Onyango et al., 2004; Rodehutscord and Dieckmann. 2005; Nyannor and Adeola, 2008; Adeola, 2010). Required for a variety of diverse biological functions, phosphorus is the second most abundant mineral element in the body following Ca (Berndt and Kumar, 2009). An inadequacy of dietary phosphorus supply would therefore negatively affect the diverse physiological processes that it is intimately involved in and hence the reduction in growth performance of ducks in each of the 3 2-week periods of d 1 to 15, d 15 to 29, and d 29 to 43, as well as the overall period from d 1 to 43. Along with calcium, between 60 and 80% of the phosphorus contained in the skeletal tissue within the body
are continuously deposited and reabsorbed from bone throughout life, thus playing key roles in mineralization of bones (Crenshaw, 2001). It is therefore appropriate to assess mineralization responses of tibia to dietary phosphorus concentration. Feeding the low-phosphorus diet to ducks predictably reduced tibia ash, and thus bone mineralization, compared with the phosphorusadequate diet. The observation that a low-phosphorus diet reduced tibia ash in ducks is also consistent with previous reports (Rodehutscord and Dieckmann, 2005; Nyannor and Adeola, 2008; Adeola, 2010). Presumably, the reduced phosphorus intake of ducks fed the lowphosphorus diet, along with calcium, decreased the synthesis and deposition of hydroxyapatite in the tibia and hence the reduction in bone mineralization. A consistent aspect of the current experiment was the improvement in growth performance, bone mineralization, and ileal digestibility and total tract retention of phosphorus with the supplementation of 500, 1,000, 1,500, or 15,000 units of phytase to each kilogram of the low-phosphorus NC diet. The diets of non-ruminant animals are supplemented with phytase to improve phytate phosphorus utilization, which consequently reduces the amount of animal waste phosphorus flowing into the environment. The improved growth performance, increased bone mineralization, and greater digestibility of phosphorus are indices of improved phosphorus utilization. The inorganic phosphorus released during hydrolysis of phytate by phytase is used by the duck to meet its phosphorus requirement. These results of the current study showed that phytase is effective in improving utilization of P in corn-soybean meal diets for ducks. Furthermore, a supra dose of phytase at 15,000 units/kg of diet did not have deleterious effects on ducks. It is instructive that the growth performance and tibia ash of ducks fed diets with phytase added at 15,000 units/kg of diet were essentially equivalent or numerically greater
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ADEOLA
than those fed the phosphorus-adequate positive control diet. Supplementing the low-phosphorus diet with 500, 1,000, 1,500, or 15,000 phytase units/kg improved weight gain during the d 1 to 43 period of the study by 26, 32, 37, or 47%, respectively, with corresponding improvements in feed intake of 15, 22, 27, or 26%. Likewise, supplementing the low-phosphorus diet with 500, 1,000, 1,500, or 15,000 phytase units/kg diet improved feed efficiency by 9, 7, 8, or 16%, respectively, during the d 1 to 43 period of the study. In a study that evaluated the efficacy of corn expressing an Escherichia coli-derived phytase gene in which the corn delivered at least 36,000 phytase units/kg diet for broiler chickens from d 7 to 21 post hatching, Nyannor and Adeola (2008) reported a 21% increase in weight gain. During the d 1 to 15 post-hatching period of the current study, adding 15,000 phytase units/kg improved weight gain by 19%. Growth performance improvements similar to those observed in the current experiment for lowphosphorus diets supplemented with between 500 and 2,000 phytase units/kg have been reported for ducks (Orban et al., 1999; Rodehutscord et al., 2003, 2006; Adeola, 2010) and broiler chickens, (Dilger et al., 2004; Pirgozliev et al., 2008). There were linear and quadratic effects of phytase addition to the low-phosphorus diet on tibia ash, the quadratic effect being greatly influenced by the higher doses of phytase. The increases in percent tibia ash were 4, 10, 10, and 15% for the respective additions of 500, 1,000, 1,500, or 15,000 phytase units/kg diet. There was an improvement in tibia of ducks from dietary supplementation of a low-phosphorus diet with phytase (Zeng et al., 2015). In earlier duck studies, Orban et al. (1999) and Adeola (2010) reported linear increases in bone mineral content, bone density, or bone ash when phytase was added to low-phosphorus diets. Augspurger et al. (2003) reported an increase in tibia ash when diets were supplemented with phytase. In other studies, Cowieson et al. (2014) and Akter et al. (2016) observed positive responses in bone ash to phytase supplementation of diets. The improvement in bone mineralization from phytase supplementation to low-phosphorus diets in the current study resulted from an increased intestinal hydrolysis of phytate to release inorganic phosphorus, as seen in the d 18 and 46 increase in ileal digestibility of phosphorus response to phytase. Supplementing the low-phosphorus diet with 500, 1,000, 1,500, or 15,000 phytase units/kg improved ileal phosphorus digestibility on d 18 by 20, 18, 33, or 61%, respectively. Furthermore, adding 500, 1,000, 1,500, or 15,000 phytase units/kg diet to the low-phosphorus diet improved ileal phosphorus digestibility by 9, 7, 8, or 16%, respectively, on d 46. These observations are consistent with previous reports in ducks (Rodehutscord et al., 2006; Adeola, 2010; Zeng et al., 2015) and broiler chickens (Nyannor and Adeola, 2008; Cowieson et al., 2014; Beeson et al., 2017). A logarithmic regression of digestible phosphorus or tibia ash against phytase intake
Figure 1. Logarithmic regression of digestible phosphorus or tibia ash against phytase intake for the 42-day duck study. Digestible phosphorus is in open squares with SEM error bars and the logarithmic fit in solid line. Tibia ash is in solid squares with SEM error bars and the logarithmic fit in dotted line.
for the 42-day study is shown in Figure 1. From the regression, supplementing the low-phosphorus diet with 500, 1,000, 1,500, or 15,000 phytase units released 1,116, 1,251, 1,330 or 1,779 mg of ileal digestible phosphorus/kg, respectively, which was used to support tibia ash. This translates to phytase-related efficiency of ileal digestible P utilization for bone mineralization of between 72 and 90%. In a study linking ileal digestible phosphorus and bone mineralization in broiler chickens fed diets supplemented with phytase, Adeola and Walk (2013) reported efficiencies between 88 and 92% for diets containing between 500 and 1,000 phytase units/kg. The low-phosphorus NC diet reduced growth performance and bone mineralization compared with the phosphorus-adequate, diet indicating inadequacy of dietary phosphorus, which showed that phosphorus was indeed a limiting nutrient in the diet. Phytase supplementation of the low-phosphorus diet consistently improved weight gain, feed intake, and feed efficiency during the d 1 to 15, 15 to 29, 29 to 43, and 1 to 43 periods of the study. Similarly, addition of phytase to the low-phosphorus diet improved utilization of phosphorus and bone mineralization. The data seem to suggest that performance of ducks on the 15,000 units/kg exceeded that of the phosphorus-adequate PC diet, which asks whether extraphosphoric effects of excess phytase dosages is a phenomenon that can be achieved in ducks as it can be with broilers. Dietary phytase at 15,000 units/kg did not have detrimental effects on growth performance, nutrient utilization, or bone mineralization of ducks, which were essentially equivalent to or numerically greater than in those fed the phosphorusadequate PC diet.
ACKNOWLEDGMENTS The author thanks Maple Leaf Farms, Leesburg, IN, for support of this research.
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PHYTASE IN DUCK DIETS
REFERENCES Adeola, O. 1999. Nutrient management procedures to enhance environmental conditions: An introduction. J. Anim. Sci. 77:427– 429. Adeola, O. 2010. Phosphorus equivalency value of an Escherichia coli phytase in the diets of White Pekin ducks. Poult. Sci. 89: 1199–1206. Adeola, O., D. Ragland, and D. King. 1997. Feeding and excreta collection techniques in metabolizable energy assays for ducks. Poult. Sci. 76:728–732. Adeola, O., and C. L. Walk. 2013. Linking ileal digestible phosphorus and bone mineralization in broiler chickens fed diets supplemented with phytase and highly soluble calcium. Poult. Sci. 92:2109–2117. AOAC International. 2000. Official Methods of Analysis. Association of Official Analytical Chemists, Arlington, VA. Akter, M., H. Graham, and P. A. Iji. 2016. Response of broiler chickens to different levels of calcium, non-phytate phosphorus and phytase. Br. Poult. Sci. 57:799–809. Augspurger, N. R., D. M. Webel, X. G. Lei, and D. H. Baker. 2003. Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs. J. Anim. Sci. 81:474–483. Beeson, L. A., C. L. Walk, M. R. Bedford, and O. A. Olukosi. 2017. Hydrolysis of phytate to its lower esters can influence the growth performance and nutrient utilization of broilers with regular or super doses of phytase. Poult. Sci. doi: 10.3382/ps/ pex012. Berndt, T., and R. Kumar. 2009. Novel mechanisms in the regulation of phosphorus homeostasis. Physiol. 24:17–25. Cowieson, A. J., F. Fru-Nji, and O. Adeola. 2014. Dietary phosphate equivalence of four forms of inorganic phosphate contrasted with a novel microbial phytase from Citrobacter braakii in broiler chickens. Anim. Prod. Sci. 55:1145–1151. dx.doi.org/10.1071/AN14489. Crenshaw, T. D. 2001. Calcium, phosphorus, vitamin D, and vitamin K in swine nutrition. Page. 187–212 in Swine Nutrition, A. J. Lewis, and L. L. Southern, ed. CRC Press, Boca Raton, FL. Dilger, R. N., E. M. Onyango, J. S. Sands, and O. Adeola. 2004. Evaluation of microbial phytase in broiler diets. Poult. Sci. 83:962– 970.
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Engelen, A. J., F. C. van der Heeft, P. H. G. Randsdorp, and E. L. C. Smit. 1994. Simple and rapid determination of phytase activity. J. AOAC Int. 77:760–764. International Union of Biochemistry. 1979. Enzyme Nomenclature: Recommendations of the Nomenclature Committee of International Union of Biochemistry. Acad. Press, New York, NY. Nolan, K. B., P. A. Duffin, and D. J. McWeeny. 1987. Effects of phytate on mineral bioavailability. In vitro studies on Mg, Ca, Fe, Cu, and Zn (also CD) solubilities in the presence of phytate. J. Sci. Food Agric. 40:79–85. Nyannor, E. K. D., and O. Adeola. 2008. Corn expressing an Escherichia coli-derived phytase gene: Comparative evaluation study in broiler chicks. Poult. Sci. 87:2015–2022. Onyango, E. M., M. R. Bedford, and O. Adeola. 2004. The yeast production system in which Escherichia Coli phytase is expressed may affect growth performance, bone ash and nutrient utilization in broiler chicks. Poult. Sci. 83:421–427. Orban, J. I., O. Adeola, and R. Stroshine. 1999. Microbial phytase in finisher diets of White Pekin ducks: Effects on growth performance, plasma phosphorus concentration, and leg bone characteristics. Poult. Sci. 78:366–377. Pirgozliev, V., O. Oduguwa, T. Acamovic, and M. R. Bedford. 2008. Effects of dietary phytase on performance and nutrient metabolism in chickens. Brit. Poult. Sci. 49:144–154. Reddy, N. R., S. K. Sathe, and D. K. Salunkhe. 1982. Phytates in legumes and cereals. Adv. Food Sci. 28:1–92. Rodehutscord, M., and A. Dieckmann. 2005. Comparative studies with three-week-old chickens, turkeys, ducks, and quails on the response in phosphorus utilization to a supplementation of monobasic calcium phosphate. Poult. Sci. 84:1252–1260. Rodehutscord, M., R. Hempel, and P. Wendt. 2003. Response of growing Pekin ducks to supplementation of monobasic calcium phosphate to low-phosphorus diets. Poult. Sci. 82:309–319. Rodehutscord, M., R. Hempel, and P. Wendt. 2006. Phytase effects on the efficiency of utilisation and blood concentrations of phosphorus and calcium in Pekin ducks. Brit. Poult. Sci. 47:311–321. SAS Institute. 2006. SAS/STAT User’s Guide. Release 9.1. (SAS Inst., Inc., Cary, NC, USA). Zeng, Q., X. Huang, Y. Luo, X. Ding, S. Bai, J. Wang, Y. Xuan, Z. Su, Y. Liu, and K. Zhang. 2015. Effects of a multi-enzyme complex on growth performance, nutrient utilization and bone mineralization of meat duck. J. Anim. Sci. Biotechnol. 6:12. doi: 10.1186/s40104-015-0013-4.
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ABSTRACT The growth performance and phosphorus utilization responses of ducks to phytase were investigated during the starter and grower phases. Fivehundred-seventy-six one-day-old drakes with an average initial BW of 55 g were grouped by BW into 8 blocks of 6 pens and assigned to 48 pens with 12 ducks per pen. The 6 dietary treatments consisted of: 1) positive control (PC), adequate in all nutrients with 4.5 g non-phytate phosphorus (nPP)/kg starter diet or 3.5 g nPP/kg grower diet; 2) negative control (NC), adequate in all other nutrients except phosphorus with 3.0 g nPP/kg starter diet or 2.0 g nPP/kg grower diet; 3) the NC plus phytase at 500 units/kg diet; 4) the NC plus phytase at 1,000 units/kg diet; 5) the NC plus phytase at 1,500 units/kg diet; and 6) the NC plus phytase at 15,000 units/kg diet. Starter and grower diets were fed from d 1 to 15 and d 15 to 43 post hatching, respectively.
Key words: bone ash, ducks, growth performance, phosphorus, phytase 2018 Poultry Science 0:1–7 http://dx.doi.org/10.3382/ps/pex352
INTRODUCTION
Ducks differ in P digestion from other avian species (Rodehutscord and Dieckmann, 2005), and a paucity of performance data exists for ducks fed phosphorusmarginal diets supplemented with phytase for an entire 42-day growth period. The objectives of the study reported here were to evaluate the growth performance, bone mineralization, as well as phosphorus utilization responses of White Pekin ducks when fed phosphorusmarginal diets supplemented with phytase for 42 days.
The phosphorus in cereal grains and oilseeds is bound in phytin, the collective term for mixed salts of magnesium, calcium, and potassium of phytic acid, the main storage form of plant phosphorus. Phytin constitutes up to 3% of many of the oilseeds and cereals used in animal feeds (Reddy et al., 1982). If hydrolyzed, phytin in feed can be a good source of phosphorus to the animal. Because intestinal phytase produced by non-ruminants is inadequate, in vivo hydrolysis of phytate is minimal, and therefore utilization of phosphorus in cereal grains and oilseed meals is limited. Consequently, much of the phytate-bound phosphorus in the diet is passed out in animal waste (Adeola, 1999). Furthermore, phytic acid has strong chelating potential in the gut and complexes Ca and Zn, thus reducing their bioavailability (Nolan et al., 1987). Adding microbial phytase to diets has been demonstrated to increase phytate hydrolysis and the availability of phosphorus in broiler chickens (Dilger et al., 2004) and ducks (Orban et al., 1999; Rodehutscord et al., 2006; Adeola, 2010).
MATERIALS AND METHODS Ducks and Diets All protocols used in the study were approved by the Purdue University Animal Care and Use Committee. Seven-hundred-sixty-eight day-old male ducks with an average initial BW of 55 g were wing-banded, weighed, and assigned to 48 pens with 16 ducks per pen. The ducks were weighed individually, sorted in decreasing order of body weight, and assigned to 6 diets such that the average initial weight of ducks was similar across dietary treatments with 16 ducks per pen and 8 pens per diet. Diets were randomly assigned to pens in each block of 6 pens. Ingredient and calculated composition of diets are presented in Table 1. The dietary treatments
C 2017 Poultry Science Association Inc. Received April 2, 2017. Accepted October 30, 2017. 1 Corresponding author: [email protected]
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ADEOLA
Table 1. Ingredient composition of the starter (d 1 to 15) and grower (d 15 to 43) experimental diets. Starter diets
Item, g/kg Corn Soybean meal Soy oil Salt Limestone Dicalcium phosphate Vitamin-mineral premix1 DL-Methionine L-Lysine.HCl Chromic oxide premix2 Phytase premix3 Phytase premix4
Positive control (PC)
Grower diets
Negative NC + NC + NC + NC + control 500 1,000 1,500 15,000 (NC) units/kg units/kg units/kg units/kg
Positive control (PC)
Negative NC + NC + NC + NC + control 500 1,000 1,500 15,000 (NC) units/kg units/kg units/kg units/kg
557.8 360.0 30.0 4.0 9.4 17.5 3.0
565.4 360.0 30.0 4.0 9.8 9.5 3.0
560.4 360.0 30.0 4.0 9.8 9.5 3.0
555.4 360.0 30.0 4.0 9.8 9.5 3.0
550.4 360.0 30.0 4.0 9.8 9.5 3.0
550.4 360.0 30.0 4.0 9.8 9.5 3.0
721.1 205.0 25.0 4.0 10.0 13.5 3.0
729.1 205.0 25.0 4.0 10.5 5.0 3.0
724.1 205.0 25.0 4.0 10.5 5.0 3.0
719.1 205.0 25.0 4.0 10.5 5.0 3.0
714.1 205.0 25.0 4.0 10.5 5.0 3.0
714.1 205.0 25.0 4.0 10.5 5.0 3.0
3.3 0.0 15.0
3.3 0.0 15.0
3.3 0.0 15.0
3.3 0.0 15.0
3.3 0.0 15.0
3.3 0.0 15.0
1.9 1.5 15.0
1.9 1.5 15.0
1.9 1.5 15.0
1.9 1.5 15.0
1.9 1.5 15.0
1.9 1.5 15.0
0.0 0.0
0.0 0.0
5.0 0.0
10.0 0.0
15.0 0.0
0.0 15.0
0.0 0.0
0.0 0.0
5.0 0.0
10.0 0.0
15.0 0.0
0.0 15.0
Total 1,000 Calculated nutrients and energy Crude protein, g/kg 220 ME, kcal/kg 3048 Ca, g/kg 8.0 Total P, g/kg 7.1 nPP, g/kg 4.5 Ca:P 1.13
1,000
1,000
1,000
1,000
1,000
1000
1,000
1,000
1,000
1,000
1,000
220 3074 6.5 5.6 3.0 1.16
220 3074 6.5 5.6 3.0 1.16
220 3074 6.5 5.6 3.0 1.16
220 3074 6.5 5.6 3.0 1.16
220 3074 6.5 5.6 3.0 1.16
160 3175 7.0 5.8 3.5 1.20
161 3202 5.5 4.3 2.0 1.28
161 3202 5.5 4.3 2.0 1.28
161 3202 5.5 4.3 2.0 1.28
161 3202 5.5 4.3 2.0 1.28
161 3202 5.5 4.3 2.0 1.28
1 Supplied the following per kg of diet: vitamin A, 5484 IU; vitamin D3 , 2643 ICU; vitamin E,11 IU; menadione sodium bisulfite, 4.38 mg; riboflavin, 5.49 mg; d-pantothenic acid, 11 mg; niacin, 44.1 mg; choline chloride, 771 mg; vitamin B12 , 13.2 μ g; biotin, 55.2 μ g; thiamine mononitrate, 2.2 mg; folic acid, 990 μ g; pyridoxine hydrochloride, 3.3 mg; I, 1.11 mg; Mn, 66.06 mg; Cu, 4.44 mg; Fe, 44.1 mg; Zn, 44.1 mg; Se, 300 μ g. 2 Prepared as 1 g chromic oxide added to 4 g of corn. 3 Phytase premix prepared by serial dilution with corn to contain 100 phytase units/g. 4 Phytase premix prepared by serial dilution with corn to contain 1,000 phytase units/g.
consisted of: 1) positive control (PC), adequate in all nutrients with 8.0 g Ca and 4.5 g non-phytate phosphorus (nPP)/kg starter diet or 7.0 g Ca and 3.5 g nPP/kg grower diet; 2) negative control (NC), adequate in all other nutrients except Ca and P with 6.5 g Ca and 3.0 g nPP/kg starter diet or 5.5 g Ca and 2.0 g nPP/kg grower diet; 3) the NC plus phytase at 500 units/kg diet; 4) the NC plus phytase at 1,000 units/kg diet; 5) the NC plus phytase at 1,500 units/kg diet; 6) the NC plus phytase at 15,000 units/kg diet. Ducks had free access to mash diets. One unit of phytase activity is defined as the quantity of enzyme required to hydrolyze 1 μmol of inorganic P/min, at pH 5.5, from an excess of 1.5 mM sodium phytate at 37◦ C (International Union of Biochemistry, 1979).
Management Flock of ducks was examined daily for any variation in appearance or behavior for 42 days. Weight gain and feed intake were monitored weekly. Each pen was equipped with one hanging cylindrical feeder, one flat plastic feed tray, one satellite waterer and a multinozzle drip nipple waterer. On d 5, the satellite water was removed from pens after ducks had been trained to drink from drip nipples; the plastic feed tray was removed at this time. A daily lighting regimen of 23 h light and 1 h dark was maintained. Room temperatures from d 1 to 8, d 8 to 15, d 15 to 22, d 22 to
29, d 29 to 35, and d 35 to 43 were kept at 35◦ C, 31◦ C, 29◦ C, 27◦ C, 24◦ C, and 22◦ C, respectively. Temperature in the room was controlled by ventilation fans. Ileal digesta and excreta were collected as previously described (Adeola et al., 1997; Adeola, 2010). Four ducks from each pen were fitted with excreta collection bags from d 15 to 18 or d 43 to 46. The collection bags were attached to the retainer rings that were sutured to the vents. The excreta collected were used to determine apparent total tract nutrient retention. On d 18 or 43, the ducks fitted with collection bags were euthanatized by carbon dioxide and intestinal digesta were collected from the Meckel’s diverticulum to the ileo-cecal junction by flushing with distilled water. Ileal digesta and excreta were stored at −20◦ C immediately after collection. Ileal digesta and excreta from ducks within a pen were pooled for the analysis of Cr, P, N, Ca, and energy. Tibia were excised from each duck and stored at −20◦ C.
Chemical and Statistical Analyses Samples for analyses were processed and analyzed as previously described by Adeola (2010). Diets, freezedried ileal digesta, and excreta were ground to pass through a 1 mm screen and dried at 105◦ C in a drying oven (Precision Scientific Co., Chicago, IL) for 24 h for dry matter determination. Gross energy was determined in a bomb calorimeter (Parr 1261 bomb
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PHYTASE IN DUCK DIETS Table 2. Analyzed composition of the experimental diets. Diets
Dry matter, %
Crude protein, %
Gross energy, kcal/g
Phytase units/kg
Ca, %
P, %
87.47 87.89 87.62 88.72 87.84 87.98
24.16 23.56 24 23.97 23.91 23.89
4.081 4.113 4.121 4.104 4.038 4.035
171 80 697 1272 1646 14,968
0.97 0.81 0.79 0.80 0.81 0.80
0.78 0.61 0.59 0.61 0.61 0.60
87.55 87.51 87.96 87.27 87.67 87.86
15.69 15.56 15.84 16.19 15.88 15.92
4.041 4.079 4.141 4.06 3.974 4.019
186 93 455 1404 1319 14,331
0.81 0.65 0.63 0.65 0.65 0.64
0.56 0.42 0.43 0.41 0.42 0.40
Starter diets Positive control (PC) Negative control (NC) NC + 500 phytase units/kg NC + 1,000 phytase units/kg NC + 1,500 phytase units/kg NC + 15,000 phytase units/kg Grower diets Positive control (PC) Negative control (NC) NC + 500 phytase units/kg NC + 1,000 phytase units/kg NC + 1,500 phytase units/kg NC + 15,000 phytase units/kg
calorimeter, Parr Instruments Co., Moline, IL) using benzoic acid as a calibration standard. The combustion method (Leco model FP-2000 N analyzer, Leco Corp., St. Joseph, MI) using EDTA as a calibration standard was employed in N analysis. Samples were digested [nitric/perchloric wet ash, AOAC 935.13 A (a)] (AOAC International, 2000) and Cr concentration was determined (Spectronic 21D, Milton Roy Company, Rochester, NY) using the method of Fenton and Fenton (1979). Total inorganic P (AOAC Official Method 985.01(A, B, D) concentration was determined by colorimetric analysis (Packard SpectraCountTM , Model # AS1000, Meriden, CT) and Ca (AOAC Official Method 985.01(A, B, D) concentration was by atomic absorption spectrophotometry (AOAC International, 2000). Phytase activity was determined by the method of Engelen et al. (1994). Tibia were defatted in a soxhlet extractor for 6 h, dried to a constant weight at 100◦ C, weighed, and then ashed in a muffle furnace at 600◦ C for 16 hours. Apparent ileal digestibility or utilization coefficient for nutrients and energy were calculated as follows: C = 1 − [(Cd/Co)
∗
(No/Nd)]
Where Cd is the concentration of Cr in the diet; Co is the concentration of Cr in the excreta or ileal digesta output; No is the concentration of nutrient or energy in the excreta or ileal digesta output; and Nd is the concentration of nutrient or energy in the diet. The product of C and the gross energy (kcal/g) concentration of the diet gives the metabolizable energy (kcal/g) or ileal digestible energy (kcal/g) of the diet. The data were analyzed using the General Linear Model procedure of SAS (SAS, 2006) in a randomized complete block design with pen as the experimental unit. Contrasts of PC vs. NC diets, and linear and quadratic contrasts were used to examine responses to supplemental graded levels of phytase at 0, 500, 1000, 1500, and 15,000 units/kg. Contrast coefficients used for unequally spaced levels were generated using the interactive matrix language procedure of SAS (2006), and statistical significance was determined at an α level of 0.05.
RESULTS The analyzed chemical composition and phytase activity of the 10 diets used in both starter and grower phases of the current study are presented in Table 2. The phytase-supplemented starter diets were formulated to contain 500, 1,000, 1500 or 15,000 units/kg analyzed at 697, 1,272, 1,646, or 14,968 units/kg, respectively. Corresponding values for the grower diets were 455, 1,404, 1,319, or 14,331 units/kg. Analyzed CP, Ca, and P were close to formulated values (Table 2). Feeding the low-phosphorus NC diet to ducks reduced (P < 0.01) gain, feed intake, and G: F compared with the PC diet, regardless of whether it was d 1 to 15, 15 to 29, 29 to 43, or 1 to 43 except for G: F during d 1 to 15 (Table 3). Supplementing the low-phosphorus NC diet with phytase resulted in both linear and quadratic improvements (P < 0.05) in gain, feed intake, and G: F. Feeding the low-P NC diet to ducks reduced (P < 0.01) tibia ash compared with the PC diet. There were both linear and quadratic increases (P < 0.05) in tibia ash with phytase supplementation (Table 3). There was no difference between the PC and NC diet in ileal digestibility except for Ca in which ileal digestibility was greater (P < 0.001) in the NC than the PC diet during the starter phase (Table 4). There were quadratic decreases (P < 0.05) in both ileal digestible energy and ileal N digestibility as phytase supplementation of the NC diet increased to 15,000 units/kg diet. Adding phytase to the NC diet linearly and quadratically increased (P < 0.001) the ileal digestibility of phosphorus. Total tract phosphorus retention and ME were lower (P < 0.05) in the NC than in the PC diet, but greater for Ca retention in NC than in the PC diet (Table 4). There was a quadratic decrease (P < 0.05) in Ca retention as phytase addition to the NC diet increased to 15,000 units/kg diet. Supplementing the lowphosphorus NC diet with phytase resulted in both linear and quadratic increases (P < 0.001) in phosphorus retention. The ileal digestibility and total tract utilization of components of the diets fed during the grower phase are presented in Table 5. Digestibility of DM, energy, N,
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ADEOLA
Table 3. Growth performance and tibia ash content of ducks fed positive control (PC), negative control (NC), and NC supplemented with phytase.1 P-value2 Phytase supplementation, Units/kg of diet Items
PC
Initial weight, g Days 1 to 15 Gain, g Feed intake, g G:F, g/kg Days 15 to 29 Gain, g Feed intake, g G:F, g/kg Days 29 to 43 Gain, g Feed intake, g G:F, g/kg Days 1 to 43 Gain, g Feed intake, g G:F, g/kg Tibia ash, %
NC
NC + 1,000
NC + 500
NC + 1,500
Phytase NC + 15,000
SEM
PC vs. NC
L
Q
1.00
1.00
0.99
55
55
55
55
55
55
0.08
551 706 781
485 584 831
527 645 816
527 662 797
544 690 788
577 702 823
11 13 13
< 0.01 < 0.01 0.01
< 0.01 < 0.01 0.40
< 0.01 < 0.01 0.01
1099 2351 467
763 1754 435
960 2106 455
997 2187 456
1045 2289 457
1089 2287 476
19 35 6
< 0.01 < 0.01 < 0.01
< 0.01 < 0.01 < 0.01
< 0.01 < 0.01 0.03
1330 4725 282
855 3887 224
1133 4130 275
1227 4515 272
1279 4682 274
1394 4606 304
34 130 9
< 0.01 < 0.01 < 0.01
< 0.01 0.02 < 0.01
< 0.01 < 0.01 < 0.01
2974 7750 384 47.5
2083 6009 348 42.5
2620 6881 381 44.3
2747 7344 374 46.7
2858 7613 376 46.9
3060 7595 403 48.8
44 137 7 1.404
< 0.01 < 0.01 < 0.01 0.02
< 0.01 < 0.01 < 0.01 0.02
< 0.01 < 0.01 0.04 0.03
1
Data are means of 8 replicate pens per diet. Probability value of the contrast of positive control vs. negative control diet (PC vs. NC), linear contrast (L), and quadratic contrast (Q). For L and Q, coefficients for unequally spaced levels were generated using Proc IML of SAS (2006). 2
Table 4. Ileal digestibility and total tract retention of ducks fed positive control (PC), negative control (NC), and NC supplemented with phytase on day 18.1 P-value2 Phytase supplementation, Units/kg of diet Items
Phytase
PC
NC
NC + 500
NC + 1,000
NC + 1,500
NC + 15,000
SEM
PC vs. NC
L
Q
69.2 70.2 3.20 76.9 44.6 42.5
69.3 69.8 3.20 78.0 40.6 57.3
70.8 71.7 3.36 78.4 48.9 59.5
67.8 67.9 3.11 75.8 47.8 59.5
68.8 68.4 3.07 75.7 54.1 60.0
68.1 68.0 3.06 75.4 65.4 54.7
1.3 1.3 0.060 1.0 2.0 3.3
0.95 0.81 0.98 0.44 0.17 < 0.001
0.40 0.28 0.04 0.11 < 0.001 0.26
0.46 0.24 0.04 0.05 < 0.001 0.52
Total tract retention DM, % 75.3 Energy, % 78.1 ME, kcal/g 3.51 N, % 65.8 P, % 44.3 Ca, % 49.4
73.7 75.1 3.29 66.4 39.4 69.0
74.8 77.1 3.500 66.9 48.6 63.3
72.4 74.1 3.29 71.6 47.3 54.3
73.0 74.7 3.34 67.1 49.4 53.0
72.4 74.9 3.36 65.7 51.8 57.9
1.7 1.5 0.065 4.3 1.5 4.8
0.50 0.16 0.02 0.92 0.03 0.01
0.55 0.77 0.99 0.66 < 0.001 0.56
0.61 0.53 0.89 0.68 < 0.001 0.01
Ileal digestibility DM, % Energy, % DE, kcal/g N, % P, % Ca, %
1
Data are means of 8 replicate cages per diet. Probability value of the contrast of positive control vs. negative control diet (PC vs. NC), linear contrast (L), and quadratic contrast (Q). For L and Q, coefficients for unequally spaced levels were generated using Proc IML of SAS (2006). 2
and P at the end of the ileum, as well as ileal digestible energy were not different between the PC and NC diets but ileal Ca digestibility was greater (P < 0.001) in the NC than in the PC diet. There were both linear and quadratic reductions (P < 0.05) in ileal digestible energy as well as a linear decrease (P = 0.05) in ileal N digestibility in response to supplementing the NC diet with 500, 1,000, 1500, or 15,000 phytase units/kg. The response (P < 0.001) in ileal phosphorus digestibility to phytase was strong, and both linear and quadratic (Table 5). There was no difference in the utilization of DM, energy, or N between ducks fed the PC or NC diets. However, ducks fed the PC diet retained more
phosphorus (P < 0.05) but less Ca (P < 0.05) than those fed the low-phosphorus NC diet. Quadratic reduction (P = 0.01) in Ca utilization with phytase supplementation of the low-phosphorus NC diet was observed. As in ileal phosphorus digestibility, there were both linear and quadratic increases (P < 0.001) in phosphorus utilization with phytase addition to the low-phosphorus NC diet (Table 5).
DISCUSSION Phytase supplementation of the diets of nonruminant animals improves the utilization of phytate
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5
PHYTASE IN DUCK DIETS
Table 5. Ileal digestibility and total tract retention of ducks fed positive control (PC), negative control (NC), and NC supplemented with phytase on day 46.1 P-value2 Phytase supplementation, FTU/kg of diet Items
Phytase
PC
NC
NC + 500
NC + 1,000
NC + 1,500
NC + 15,000
SEM
PC vs. NC
L
Q
70.6 71.7 3.27 78.3 45.5 43.4
70.7 71.2 3.26 79.6 40.4 58.4
72.2 73.1 3.43 80.1 49.9 60.7
69.3 69.3 3.18 77.6 50.3 60.7
70.4 70.0 3.14 77.6 56.4 61.4
69.5 69.5 3.13 77.4 66.8 55.9
1.33 1.32 0.061 1.02 2.04 3.35
0.97 0.83 1.00 0.45 0.15 < 0.001
0.41 0.29 0.04 0.11 < 0.001 0.27
0.47 0.24 0.04 0.05 < 0.001 0.53
Total tract retention DM, % 77.2 Energy, % 79.8 ME, kcal/g 3.61 N, % 67.6 P, % 45.2 Ca, % 50.4
75.2 76.6 3.42 67.7 38.7 70.4
76.5 78.0 3.51 68.4 49.6 64.7
74.0 75.7 3.43 73.0 48.7 55.4
74.7 76.9 3.41 68.8 50.9 54.2
73.9 76.5 3.44 66.8 52.9 59.3
1.74 1.52 0.066 4.39 1.53 4.92
0.56 0.79 1.01 0.67 < 0.001 0.57
0.62 0.54 0.91 0.69 < 0.001 0.01
Ileal digestibility DM, % Energy, % DE, kcal/g N, % P, % Ca, %
0.51 0.16 0.02 0.94 0.03 < .0001
1
Data are means of 8 replicate cages per diet. Probability value of the contrast of positive control vs. negative control diet (PC vs. NC), linear contrast (L), and quadratic contrast (Q). For L and Q, coefficients for unequally spaced levels were generated using Proc IML of SAS (2006). 2
phosphorus and ultimately reduces the amount of phosphorus flowing into the environment from animal waste. Results of studies abound in the literature on phytase supplementation of the diets of broiler chickens from day-old to market weight. However the same cannot be said of ducks for which results of only a few studies have been published but for various stages of the life cycle, such as parts of the starter period or portions of the grower period, and not for the entire life cycle from day-old to market weight. In the current experiment, growth performance responses of ducks for a 42-day life cycle and nutrient utilization responses to phytase supplementation were investigated. In each of the 3 2-week periods of d 1 to 15, d 15 to 29, and d 29 to 43, as well as the overall period of d 1 to 43, ducks responded to the phosphorus-adequate diets with improvements in weight gain, feed intake, and feed efficiency when compared with the low-phosphorus diets. This is a clear indication that phosphorus was indeed a limiting nutrient for the growth performance of ducks used in the current study. This observation is consistent with previous reports of low-phosphorusinduced reduction in growth performance of ducks and broiler chickens (Orban et al., 1999; Dilger et al., 2004; Onyango et al., 2004; Rodehutscord and Dieckmann. 2005; Nyannor and Adeola, 2008; Adeola, 2010). Required for a variety of diverse biological functions, phosphorus is the second most abundant mineral element in the body following Ca (Berndt and Kumar, 2009). An inadequacy of dietary phosphorus supply would therefore negatively affect the diverse physiological processes that it is intimately involved in and hence the reduction in growth performance of ducks in each of the 3 2-week periods of d 1 to 15, d 15 to 29, and d 29 to 43, as well as the overall period from d 1 to 43. Along with calcium, between 60 and 80% of the phosphorus contained in the skeletal tissue within the body
are continuously deposited and reabsorbed from bone throughout life, thus playing key roles in mineralization of bones (Crenshaw, 2001). It is therefore appropriate to assess mineralization responses of tibia to dietary phosphorus concentration. Feeding the low-phosphorus diet to ducks predictably reduced tibia ash, and thus bone mineralization, compared with the phosphorusadequate diet. The observation that a low-phosphorus diet reduced tibia ash in ducks is also consistent with previous reports (Rodehutscord and Dieckmann, 2005; Nyannor and Adeola, 2008; Adeola, 2010). Presumably, the reduced phosphorus intake of ducks fed the lowphosphorus diet, along with calcium, decreased the synthesis and deposition of hydroxyapatite in the tibia and hence the reduction in bone mineralization. A consistent aspect of the current experiment was the improvement in growth performance, bone mineralization, and ileal digestibility and total tract retention of phosphorus with the supplementation of 500, 1,000, 1,500, or 15,000 units of phytase to each kilogram of the low-phosphorus NC diet. The diets of non-ruminant animals are supplemented with phytase to improve phytate phosphorus utilization, which consequently reduces the amount of animal waste phosphorus flowing into the environment. The improved growth performance, increased bone mineralization, and greater digestibility of phosphorus are indices of improved phosphorus utilization. The inorganic phosphorus released during hydrolysis of phytate by phytase is used by the duck to meet its phosphorus requirement. These results of the current study showed that phytase is effective in improving utilization of P in corn-soybean meal diets for ducks. Furthermore, a supra dose of phytase at 15,000 units/kg of diet did not have deleterious effects on ducks. It is instructive that the growth performance and tibia ash of ducks fed diets with phytase added at 15,000 units/kg of diet were essentially equivalent or numerically greater
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6
ADEOLA
than those fed the phosphorus-adequate positive control diet. Supplementing the low-phosphorus diet with 500, 1,000, 1,500, or 15,000 phytase units/kg improved weight gain during the d 1 to 43 period of the study by 26, 32, 37, or 47%, respectively, with corresponding improvements in feed intake of 15, 22, 27, or 26%. Likewise, supplementing the low-phosphorus diet with 500, 1,000, 1,500, or 15,000 phytase units/kg diet improved feed efficiency by 9, 7, 8, or 16%, respectively, during the d 1 to 43 period of the study. In a study that evaluated the efficacy of corn expressing an Escherichia coli-derived phytase gene in which the corn delivered at least 36,000 phytase units/kg diet for broiler chickens from d 7 to 21 post hatching, Nyannor and Adeola (2008) reported a 21% increase in weight gain. During the d 1 to 15 post-hatching period of the current study, adding 15,000 phytase units/kg improved weight gain by 19%. Growth performance improvements similar to those observed in the current experiment for lowphosphorus diets supplemented with between 500 and 2,000 phytase units/kg have been reported for ducks (Orban et al., 1999; Rodehutscord et al., 2003, 2006; Adeola, 2010) and broiler chickens, (Dilger et al., 2004; Pirgozliev et al., 2008). There were linear and quadratic effects of phytase addition to the low-phosphorus diet on tibia ash, the quadratic effect being greatly influenced by the higher doses of phytase. The increases in percent tibia ash were 4, 10, 10, and 15% for the respective additions of 500, 1,000, 1,500, or 15,000 phytase units/kg diet. There was an improvement in tibia of ducks from dietary supplementation of a low-phosphorus diet with phytase (Zeng et al., 2015). In earlier duck studies, Orban et al. (1999) and Adeola (2010) reported linear increases in bone mineral content, bone density, or bone ash when phytase was added to low-phosphorus diets. Augspurger et al. (2003) reported an increase in tibia ash when diets were supplemented with phytase. In other studies, Cowieson et al. (2014) and Akter et al. (2016) observed positive responses in bone ash to phytase supplementation of diets. The improvement in bone mineralization from phytase supplementation to low-phosphorus diets in the current study resulted from an increased intestinal hydrolysis of phytate to release inorganic phosphorus, as seen in the d 18 and 46 increase in ileal digestibility of phosphorus response to phytase. Supplementing the low-phosphorus diet with 500, 1,000, 1,500, or 15,000 phytase units/kg improved ileal phosphorus digestibility on d 18 by 20, 18, 33, or 61%, respectively. Furthermore, adding 500, 1,000, 1,500, or 15,000 phytase units/kg diet to the low-phosphorus diet improved ileal phosphorus digestibility by 9, 7, 8, or 16%, respectively, on d 46. These observations are consistent with previous reports in ducks (Rodehutscord et al., 2006; Adeola, 2010; Zeng et al., 2015) and broiler chickens (Nyannor and Adeola, 2008; Cowieson et al., 2014; Beeson et al., 2017). A logarithmic regression of digestible phosphorus or tibia ash against phytase intake
Figure 1. Logarithmic regression of digestible phosphorus or tibia ash against phytase intake for the 42-day duck study. Digestible phosphorus is in open squares with SEM error bars and the logarithmic fit in solid line. Tibia ash is in solid squares with SEM error bars and the logarithmic fit in dotted line.
for the 42-day study is shown in Figure 1. From the regression, supplementing the low-phosphorus diet with 500, 1,000, 1,500, or 15,000 phytase units released 1,116, 1,251, 1,330 or 1,779 mg of ileal digestible phosphorus/kg, respectively, which was used to support tibia ash. This translates to phytase-related efficiency of ileal digestible P utilization for bone mineralization of between 72 and 90%. In a study linking ileal digestible phosphorus and bone mineralization in broiler chickens fed diets supplemented with phytase, Adeola and Walk (2013) reported efficiencies between 88 and 92% for diets containing between 500 and 1,000 phytase units/kg. The low-phosphorus NC diet reduced growth performance and bone mineralization compared with the phosphorus-adequate, diet indicating inadequacy of dietary phosphorus, which showed that phosphorus was indeed a limiting nutrient in the diet. Phytase supplementation of the low-phosphorus diet consistently improved weight gain, feed intake, and feed efficiency during the d 1 to 15, 15 to 29, 29 to 43, and 1 to 43 periods of the study. Similarly, addition of phytase to the low-phosphorus diet improved utilization of phosphorus and bone mineralization. The data seem to suggest that performance of ducks on the 15,000 units/kg exceeded that of the phosphorus-adequate PC diet, which asks whether extraphosphoric effects of excess phytase dosages is a phenomenon that can be achieved in ducks as it can be with broilers. Dietary phytase at 15,000 units/kg did not have detrimental effects on growth performance, nutrient utilization, or bone mineralization of ducks, which were essentially equivalent to or numerically greater than in those fed the phosphorusadequate PC diet.
ACKNOWLEDGMENTS The author thanks Maple Leaf Farms, Leesburg, IN, for support of this research.
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