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Influence of limestone and phytase on broiler performance, gastrointestinal pH, and apparent ileal nutrient digestibility
Influence of limestone and phytase on broiler performance, gastrointestinal pH, and apparent ileal nutrient digestibility C. L. Walk,*1 M. R. Bedford,* and A. P. McElroy† *AB Vista Feed Ingredients, Marlborough, Wiltshire, SN8 4AN, United Kingdom; and †Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg 24601 Ca. Phytase at 5,000 FTU/kg increased (P < 0.05) pH in the gizzard, duodenum, jejunum, and ileum. Apparent ileal P digestibility was increased (P < 0.05) in broilers fed 0.64% Ca compared with broilers fed 1.03% Ca (0.68 vs. 0.73, respectively). Apparent ileal Ca digestibility was increased (P < 0.05) in broilers fed 1.03% Ca compared with broilers fed 0.64% Ca (0.67 vs. 0.53, respectively). Phytase improved AID of CP in broilers fed 1.0% Ca but did not have an effect on AID of CP in broilers fed 0.64% Ca, which resulted in a Ca × phytase interaction (P < 0.05). In conclusion, high dietary Ca increased pH in gizzard and ileum and interfered with the AID of P and CP. The interactions between Ca and phytase in the gastrointestinal tract are complex, and feeding phytase at doses above industry recommendations may allow for reduced-Ca diets while maintaining broiler performance, bone ash, and improving amino acid digestibility.
Key words: broiler, calcium, phosphorus, phytase 2012 Poultry Science 91:1371–1378 http://dx.doi.org/10.3382/ps.2011-01928
INTRODUCTION The amount of Ca required to sustain the broilers’ physiological requirement may vary with age, Ca source, and the phytate content of the diet. For example, if a normal corn-soybean meal diet contains 1% phytate, which can chelate 0.36% Ca (Nelson et al., 1968), dietary phytase may liberate up to 0.36% Ca with complete phytate hydrolysis. Shirley and Edwards (2003) reported 12,000 U of phytase/kg of diet hydrolyzed 95% of the phytate phosphorus in corn and soybean meal diets. Thus, complete phytate hydrolysis may allow for significant reductions in dietary Ca without influencing broiler performance. High dietary Ca has been implicated in reduced phytase efficacy (Ballam et al., 1984; Tamim and Angel, 2003; Tamim et al., 2004), reduced animal performance ©2012 Poultry Science Association Inc. Received October 9, 2011. Accepted February 29, 2012. 1 Corresponding author: [email protected]
(Sebastian et al., 1996; Powell et al., 2011), increased gastrointestinal pH (Guinotte et al., 1995), and interference with macromineral absorption (Hurwitz et al., 1978; Simpson and Wise, 1990). Therefore, reducing dietary Ca and removing Ca chelators, such as phytate, by supplementing high levels of dietary phytase may improve the bird’s utilization efficiency of dietary Ca and P. Reductions in dietary Ca may also indirectly improve amino acid (AA) utilization via a reduction in gastric pH and improved pepsin efficacy. Chicken pepsin activity is pH dependent, with an optimum activity at pH 2.8 (Bohak, 1969), and as a consequence, high dietary Ca may result in unfavorable pH in the proventriculus/gizzard. An increase in gizzard pH significantly reduced Ca solubility in broilers (Guinotte et al., 1995), and a higher pH has been implicated in Ca-phytate interactions in the gastrointestinal tract, as reviewed by Selle et al. (2009). Phytase may spare excess pepsin secretion by hydrolyzing phytate and alleviating the negative effect of phytate on gastric protein hydrolysis and resultant AA digestibility (Cowieson and Ravindran, 2007). High dietary Ca may influence
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ABSTRACT An experiment was conducted to determine the influence of 2 levels of dietary Ca from limestone and 3 levels of phytase on broiler performance, bone ash, gastrointestinal pH, and apparent ileal digestibility (AID) of Ca, P, and amino acids. Cobb 500 broilers (n = 576) were allowed access to one of 6 cornsoy diets from 0 to 16 d. Experimental diets contained 1.03% or 0.64% Ca from limestone and 0.61% total P. Each diet was supplemented with 0, 500, or 5,000 FTU/kg of phytase to create a 2 × 3 factorial experiment. Broiler feed intake (FI) and BW gain were not affected by dietary Ca or phytase. Feed conversion ratio was improved (P < 0.05) as dietary phytase increased (1.36, 1.34, and 1.31, respectively). Tibia ash percent was reduced (P < 0.05) from 41.4 to 40.0% as dietary Ca decreased but increased with phytase addition (P < 0.05). Gizzard and ileal pH were reduced (P < 0.05) in broilers fed 0.64% Ca compared with broilers fed 1.03%
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gastrointestinal pH and directly reduce protein digestion through reduced pepsin efficacy or indirectly influence Ca-phytate precipitation. Dietary phytase may hydrolyze phytate, reducing Ca-phytate interactions, and allow for reduced dietary Ca from limestone while maintaining broiler performance. Reduced dietary Ca in the presence of phytase may also spare pepsin efficacy and improve AA digestibility through a reduction in proximal gastric pH. The objectives of this trial were to determine the influence of 3 levels of phytase in low or high Ca diets on broiler performance, gastrointestinal pH, tibia ash, and apparent ileal digestibility (AID) of Ca, P, and AA.
MATERIALS AND METHODS Birds and Husbandry
Dietary Treatments Experimental diets were arranged as a 2 × 3 factorial including 2 levels of Ca (1.03 and 0.64%) and 3 levels of phytase (0, 500, and 5,000 FTU/kg; AB Vista Feed Ingredients, Marlborough, UK). This resulted in a total of 6 treatment groups replicated by 16 pens of 6 chicks each (96 chicks/dietary treatment). Diets were fed in mash form and based on corn and soybean meal to meet or exceed Cobb nutrient requirements according to the Cobb 500 Broiler Performance and Nutrient Supplement (2008; Siloam Springs, Arkansas) with the exception of Ca and available P (Table 1). Corn was added in place of phytase, and titanium dioxide was added as an inert marker for nutrient digestibility evaluation. The phytase used in the experiment was an Escherichia coli 6-phytase expressed in Pichia pastoris with an expected activity of 5,000 FTU/g. 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. All experimental procedures were approved by the Virginia Tech Institutional Animal Care and Use Committee.
Response Variables Birds were weighed by pen before placement (d 0) and on d 16 to measure mean treatment BW and calculate mean BW gain. Feed intake (FI) was also measured on d 0 and d 16 and used to calculate feed conversion (FCR). Mortality was recorded daily, and any
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Cobb 500, male broilers (576) were obtained from a commercial hatchery at day of hatch and transported to the research facilities at Virginia Tech. On arrival, chicks were randomized, weighed by pen, and placed in Petersime battery brooders with respect to 6 dietary treatments. Birds were maintained on a constant lighting program and allowed ad libitum access to the treatment diets for the duration of the trial (d 0 to 16).
birds culled or dead were weighed. Treatment FI and FCR were adjusted according to the number of bird days, which is defined as the number of bird days alive in each cage multiplied by the number of days without incidence of mortality. Birds for sampling were stunned by exposure to CO2 gas for approximately 30 s and euthanized by cervical dislocation, for collection of tibias and digesta. On d 16, 2 birds/pen of average BW were killed to obtain measurements of pH from the proximal gizzard (proventricular opening), distal gizzard (duodenal opening), duodenum (duodenum loop), medial jejunum (defined as the intestinal section distal to the duodenal loop and proximal to the Meckel’s diverticulum), proximal ileum (approximately 5 cm distal to the Meckel’s diverticulum), and the distal ileum (approximately 2 cm proximal to the ileocecal junction). From each bird, pH measurements were obtained directly from the digesta contents in the lumen using a digital pH meter (Mettler-Toledo, Columbus, OH) and spear-tip electrode (Sensorex, Garden Grove, CA). The pH values from each gastrointestinal section/bird were then pooled to obtain a mean pH value/pen. Left tibias were obtained from 3 birds/pen on d 16 and pooled for determination of bone ash. Tibias were stripped of adhering tissues, wrapped in cheesecloth, and dried overnight at 100°C. Fat was extracted from the tibias using a Soxhelt apparatus and 100% ethyl ether according to modified methods of Watson et al. (2006). Fat-extracted tibias were dried for 24 h at 100°C and ashed in a muffle furnace for 24 h at 600°C to determine bone ash. Digesta was collected from the ileum, defined as the Meckel’s diverticulum to the ileocecal junction, from the 5 birds/pen euthanized to measure pH and tibias. The collected digesta was pooled by pen, weighed, and frozen at −20°C until further chemical analysis. Prior to analysis, digesta samples were lyophilized and ground to pass a 1-mm screen. Amino acid concentrations in the diet and ileal digesta were determined by HPLC according to methods described by the AOAC (2006) for standard protein hydrolysis (method 45.3.05). Diets and ileal digesta were subjected to a total Ca and P analysis using a nitric acid and perchloric acid wet ash digestion. Phosphorus in the diets and digesta was analyzed colorimetrically using a Genesys 5 spectrophotometer (Thermo Electron Corp., Madison, WI) at 410 nm according to methods of the AOAC (1970) for P (method 7.095–7.098). Calcium in the diets and digesta was obtained using a Perkin Elmer Atomic Absorption spectrophotometer (Analyst 800, Ueberlingen, Germany) and 1% lanthanum oxide according to Analyst 800 Atomic Absorption Spectrophotometer User’s Guide (Perkin Elmer Insruments, 1998) methods. Phytase recovered in the diets was analyzed by Enzyme Services and Consultancy (Blackwood, Wales) according to modified methods of Engelen et al. (2001). Titanium dioxide in the diet and ileal digesta was analyzed ac-
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LIMESTONE AND PHYTASE Table 1. Composition and nutrient content of experiment diets Item (%)
Low Ca
59.85 29.16 4.00 2.50 1.36 0.77 1.21 0.33 0.16 0.23 0.10 0.10 0.10 0.01 0.01 0.10 87.29 21.00 1.20 0.80 0.89 0.90 0.53 0.32 3,003 21.79 1.36 0.82 1.03 0.61
61.51 28.88 4.00 2.50 0.77 0.77 0.43 0.33 0.17 0.23 0.10 0.10 0.10 0.01 0.01 0.10 87.10 21.00 1.20 0.80 0.89 0.60 0.53 0.32 3,003 22.39 1.37 0.82 0.64 0.61
1Dicalcium
phosphate supplied 18.5% P and 22% Ca. supplied 38% Ca. 3Supplied per kilogram of diet: iron (ferrous sulfate), 40 mg; manganese (manganese sulfate and manganous oxide), 120 mg; zinc (zinc oxide), 210 mg; cobalt (cobalt carbonate), 2.2 mg; iodine (calcium iodate), 132 mg. 4Supplied per kilogram of diet: vitamin A, 8,818 IU; vitamin D , 2,646 ICU; vitamin E, 22 IU; vitamin B , 26 3 12 μg; riboflavin, 8.8 mg; niacin, 88 mg; d-pantothenic acid, 22 mg; vitamin K, 2.6 mg; folic acid, 2.2 mg; vitamin B6, 4.3 mg; thiamine, 3.7 mg; d-biotin, 220 μg. 5Supplied per kilogram of diet: 600 ppm. 6Corn was supplemented in place of phytase in diets containing 0 or 500 FTU/kg of phytase to equal 100%. 2Limestone
cording to methods of Short et al. (1996). Diet and ileal titanium dioxide, AA, Ca, and P values were used to calculate AID using the following equation: Digestibility coefficient = [(nutrient/TiO2)diet – (nutrient/TiO2)ileum]/(nutrient/TiO2)diet.
Statistical Analysis Data was analyzed as a 2 × 3 factorial using the fit model platform of JMP 8.0 (SAS Institue Inc., Cary, NC). Pen served as the experimental unit for all parameters measured. The statistical model included Ca, phytase, and the interaction of Ca × phytase. When differences were significant, means were separated using Tukey’s honestly significant difference test. Significance was accepted at P ≤ 0.05. In the event that interactions were not significant, main effects were discussed.
RESULTS The analyzed dietary Ca and P were within acceptable ranges and in agreement with formulated values when mixing and assay errors were considered (Table 1). Phytase activities recovered in the diets were approximately 37% higher than formulated, and these results may be associated with an actual phytase activity higher than expected, sample variation and mixing, and assay errors. Feed intake and BW gain were not influenced by dietary Ca, phytase, or the interaction of Ca × phytase (Table 2). Phytase supplementation at 500 FTU/kg improved (P ≤ 0.05) FCR compared with broilers fed 0 FTU/kg, and supplementation at 5,000 FTU/kg improved (P ≤ 0.05) FCR compared with broilers fed 0 or 500 FTU/kg (1.36, 1.34, and 1.31, respectively; Table 2). Feed conversion was not influenced by dietary Ca or the interaction of Ca × phytase. Overall, experimental mortality was low (3.41%), but
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Ingredient Corn Soybean meal, 45% Dried distillers grains with solubles, 26% Poultry by-product, 58% Poultry fat Dicalcium phosphate1 Limestone2 Salt l-Lysine HCl dl-Methionine Titanium dioxide Trace mineral premix3 Vitamin premix4 l-Threonine Selenium premix5 Phytase6 Calculated composition DM CP Lysine Threonine Total sulfur amino acids Ca Total P Available P Nutrient composition Energy (ME; kcal/kg) Analyzed composition CP Lysine Threonine Ca Total P
High Ca
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Table 2. Influence of Ca and phytase on growth performance, mortality, and tibia ash of broilers1 from d 0 to 16 Dietary treatment Ca (%)
Phytase (FTU)
1.03 0.64
BW gain (g)
FCR2
Mortality (%)
Tibia ash (%)
626.9 629.2 621.1 634.1 623.6 616.6 7.679 625.7 624.8 4.433 630.5 626.4 618.8 5.430 0.8778 0.3100 0.6565
462.3 474.8 476.3 468.3 463.3 472.1 5.627 471.1 467.9 3.249 465.3 469.1 474.2 3.979 0.4843 0.2890 0.3053
1.356 1.327 1.305 1.354 1.346 1.306 0.0085 1.329 1.335 0.0049 1.355a 1.337b 1.306c 0.0060 0.3877 0.0001 0.3938
1.04 2.08 4.17 0.00 4.17 8.33 2.43 4.17 0.52b 3.13ab 6.25a 0.2590 0.0059 0.3664
40.07 41.50 42.72 39.19 39.93 40.80 0.2455 41.43a 39.97b 0.1417 39.63c 40.71b 41.76a 0.1736 0.0001 0.0001 0.1029
a–cMeans
within the same column with no common superscript differ significantly (P ≤ 0.05). represent the average response of 16 replicate pens (96 chicks)/treatment. 2FCR = feed conversion ratio, feed:gain; corrected for mortality. 1Means
broilers fed 5,000 FTU/kg of phytase had an increase (P ≤ 0.05) in percent mortality compared with broilers fed 0 FTU/kg (6.25 vs. 0.52%, respectively; Table 2). Tibia ash was not influenced by the interaction of Ca × phytase (Table 2). Reducing dietary Ca from 1.03% to 0.64% reduced (P ≤ 0.05) broiler tibia ash (41.4 and 40.0%, respectively). Phytase supplementation at 500 FTU/kg increased (P ≤ 0.05) tibia ash compared with broilers fed 0 FTU/kg phytase. Broilers fed 5,000 FTU/kg phytase had increased (P ≤ 0.05) tibia ash compared with broilers fed 500 FTU/kg. Gastrointestinal pH was not influenced by Ca × phytase interaction (Table 3). Distal gizzard and distal ileal pH was lower (P ≤ 0.05) in broilers fed 0.64% Ca compared with broilers fed 1.03% Ca. Supplementing broiler diets with 5,000 FTU/kg of phytase increased (P ≤ 0.05) pH in the gastrointestinal tract compared with broilers fed 0 or 500 FTU/kg of phytase, except in the distal gizzard where 5,000 FTU/kg resulted in higher pH compared with 500 FTU/kg of supplementation, but neither of these were different from broilers fed 0 FTU/kg of phytase. Apparent ileal P or Ca digestibility was not influenced by a Ca × phytase interaction or by phytase supplementation (Table 4). Reducing dietary Ca from 1.03% to 0.64% improved (P ≤ 0.05) AID of P approximately 7%. Apparent ileal Ca digestibility was reduced (P ≤ 0.05) approximately 21% in broilers fed 0.64% Ca compared with broilers fed 1.03% Ca. Apparent ileal serine, tyrosine, and total CP digestibility were influenced (P ≤ 0.05) by a Ca × phytase interaction (Tables 5 and 6). In general, increasing phytase improved AID of serine, tyrosine, or total CP, when broilers were fed 1.03% Ca. However, when broil-
ers were fed reduced (0.64%) Ca diets, phytase supplementation did not influence or negatively influenced AID of serine, tyrosine, or total CP, which resulted in a Ca × phytase interaction. There were no other Ca × phytase interactions influencing AID of AA. However, the AID of all AA was influenced by the main effect of phytase, with the exception of threonine or histidine (Tables 5 and 6). In general, phytase supplementation at 5,000 FTU/kg improved (P ≤ 0.05) the AID of AA approximately 2 to 4% compared with phytase at 0 or 500 FTU/kg. The main effect of Ca did not influence AID of AA.
DISCUSSION Broiler FI or BW gain was not influenced by dietary Ca or phytase, which would suggest Ca or P were not limiting growth performance. Sebastian et al. (1996) reported no differences in FI or BW gain when 21-d-old broilers were fed corn-soy diets formulated to contain 0.60% or 1.0% Ca. However, Augspurger and Baker (2004b) observed a linear improvement in BW gain as dietary Ca increased from 0 to 1.0%. In addition, phytase supplementation improved BW gain in low Ca diets (Sebastian et al., 1996; Augspurger and Baker, 2004b; Powell et al., 2011), suggesting phytase may improve Ca utilization by hydrolyzing phytate. Although the overall mortality in the experiment was low (3.41%), the increase in mortality associated with 5,000 FTU/kg of phytase was not expected. The use of a small population of birds in the experiment, necropsy of the dead birds, and growth performance and bone ash of the remaining birds would indicate mortality was not the result of any negative biological function of
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SEM Ca 1.03 0.64 SEM Phytase 0 500 5,000 SEM P-value Ca Phytase Ca × phytase
0 500 5,000 0 500 5,000
Feed intake (g)
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LIMESTONE AND PHYTASE Table 3. Influence of Ca and phytase on
broiler1
Dietary treatment Ca (%)
Gizzard
Duodenum
Jejunum
Ileum
Proximal
Distal
Loop
Medial
Proximal
Distal
0 500 5,000 0 500 5,000
1.94 1.84 2.18 1.85 1.76 2.09 0.075 1.98 1.90 0.043 1.89b 1.80b 2.13a 0.053 0.1707 0.0001 0.9921
2.40 2.54 2.63 2.44 2.15 2.53 0.090 2.52a 2.37b 0.052 2.42ab 2.34b 2.58a 0.064 0.0409 0.0330 0.0574
6.22 6.18 6.26 6.14 6.17 6.26 0.029 6.22 6.19 0.017 6.18b 6.17b 6.26a 0.020 0.1672 0.0066 0.3541
5.90 5.96 6.01 5.91 5.86 6.00 0.028 5.95 5.92 0.016 5.90b 5.91b 6.00a 0.019 0.1831 0.0008 0.1189
5.86 5.90 5.97 5.87 5.85 5.91 0.030 5.91 5.88 0.017 5.86b 5.88b 5.94a 0.021 0.1723 0.0276 0.4356
6.63 6.74 7.06 6.49 6.55 6.92 0.078 6.81a 6.65b 0.045 6.56b 6.65b 6.99a 0.055 0.0165 0.0001 0.9255
0.64
a,bMeans 1Means
within the same column with no common superscript differ significantly (P ≤ 0.05). represent the average response of 16 replicate pens (32 chicks)/treatment.
phytase but rather the loss of 2 birds in one pen that equaled 33% mortality for that particular replicate. Increasing dietary phytase significantly improved broiler FCR and bone ash, and this has been previously reported in broilers fed diets containing supplemental phytase in excess of 1,000 FTU/kg (Shirley and Edwards, 2003; Augspurger and Baker, 2004a). Reducing dietary Ca significantly reduced broiler tibia ash, even though broiler growth performance was not influenced by Ca level, which would suggest that Ca was limiting for this parameter and that tibia ash is a more
sensitive indicator of mineral status than growth performance. Percentage of tibia ash tended to decrease in broilers fed 0.60% Ca compared with broilers fed 1.0% Ca (Sebastian et al., 1996). However, Powell et al. (2011) reported an improvement in percentage of tibia ash as dietary Ca was reduced from 1.33% to 0.67%. The differences in the published literature may be related to the level of P in the experimental diets and the Ca:P ratio. For example, Powell et al. (2011) fed 0.20% nonphytate phosphorus, whereas Sebastian et al. (1996) fed 0.31% available P, which was closer to the
Table 4. Influence of Ca and phytase on apparent ileal Ca and P digestibility of 16-d-old broilers1 Apparent ileal digestibility (digestibility coefficient)
Dietary treatment Ca (%) 1.03 0.64 SEM Ca 1.03 0.64 SEM Phytase 0 500 5,000 SEM P-value Ca Phytase Ca × phytase a,bMeans 1Means
Phytase (FTU)
P
Ca
0 500 5,000 0 500 5,000
0.69 0.68 0.67 0.75 0.71 0.73 0.0299 0.68b 0.73a 0.0173 0.72 0.69 0.70 0.0217 0.0410 0.6933 0.9375
0.66 0.67 0.69 0.52 0.56 0.51 0.0454 0.67a 0.53b 0.0264 0.59 0.61 0.60 0.0322 0.0004 0.8447 0.7583
within the same column with no common superscript differ significantly (P ≤ 0.05). represent the average response of 16 replicate pens (64 chicks)/treatment.
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Phytase (FTU)
1.03
SEM Ca 1.03 0.64 SEM Phytase 0 500 5,000 SEM P-value Ca Phytase Ca × phytase
gastrointestinal pH from d 0 to 16
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Table 5. Influence of Ca and phytase on apparent ileal essential amino acid digestibility of 16-d-old broilers1 Dietary treatment Phytase (FTU)
Ca (%) 1.03
0 500 5,000 0 500 5,000
0.64
Thr
Arg
Val
Iso
Leu
Phe
Lys
His
0.75 0.76 0.78 0.77 0.75 0.77 0.009 0.76 0.76 0.005 0.76 0.76 0.77 0.007 0.7753 0.2072 0.1360
0.87 0.89 0.90 0.89 0.88 0.90 0.004 0.89 0.89 0.003 0.88b 0.88b 0.90a 0.003 0.3967 0.0001 0.0574
0.79 0.81 0.83 0.80 0.80 0.82 0.008 0.81 0.81 0.004 0.79b 0.81b 0.82a 0.005 0.8670 0.0028 0.2248
0.81 0.83 0.85 0.83 0.83 0.85 0.007 0.83 0.84 0.004 0.82b 0.83b 0.85a 0.005 0.6737 0.0003 0.2848
0.83 0.85 0.86 0.84 0.84 0.85 0.006 0.85 0.84 0.004 0.84b 0.84b 0.86a 0.004 0.9045 0.0032 0.0790
0.83 0.85 0.87 0.84 0.84 0.86 0.006 0.85 0.85 0.004 0.84b 0.84b 0.86a 0.004 0.8964 0.0001 0.0980
0.86 0.87 0.89 0.88 0.88 0.89 0.005 0.87 0.88 0.003 0.87b 0.87b 0.89a 0.004 0.1232 0.0010 0.2166
0.84 0.85 0.86 0.85 0.85 0.85 0.006 0.85 0.85 0.003 0.84 0.85 0.86 0.004 0.9865 0.1317 0.1336
a,bMeans 1Means
within the same column with no common superscript differ significantly (P ≤ 0.05). represent the average response of 8 replicate pens (32 chicks)/treatment.
level of available P fed in the current experiment. When evaluating the influence of Ca:P ratio on broiler weight gain or toe ash, Qian et al. (1997) reported a 24% or 15% improvement, respectively, in broilers fed low Ca diets with low P (1.1:1, Ca:P ratio) compared with broilers fed high Ca diets with low P (2.0:1 Ca:P ratio). Gizzard and ileal pH were influenced by Ca, indicating high dietary limestone may act as an antacid in the distal portions of the gizzard and ileum. Increasing dietary limestone increased gizzard pH of immature pullets (Guinotte et al., 1995) and increased crop and ileal
pH in 12-d-old broilers (Shafey et al., 1991), but it did not influence gizzard or small intestinal pH in mature laying hens (Guinotte et al., 1995; Gordon and Roland, 1997). Conflicting reports regarding differences in gastrointestinal pH and limestone supplementation may arise from the large variation in gastrointestinal pH between broilers. For example, in the current experiment, the pH in the gizzard or ileum of broilers ranged from approximately 1.0 to 4.5 and 4.5 to 7.5, respectively. Large variation in pH of the specific digestive organs of broilers may confound or mask differences in pH asso-
Table 6. Influence of Ca and phytase on apparent ileal nonessential amino acid digestibility of 16-d-old broilers1 Dietary treatment Ca (%)
Phytase (FTU)
1.03 0.64 SEM Ca 1.03 0.64 SEM Phytase 0 500 5,000 SEM P-value Ca Phytase Ca × phytase a–cMeans 1Means
0 500 5,000 0 500 5,000
Apparent ileal digestibility coefficient Asp
Ser
0.79 0.81 0.83 0.81 0.80 0.83 0.007
0.79bc 0.80abc 0.81a 0.81a 0.78c 0.81ab
Glu
Pro
0.008
0.86 0.88 0.90 0.88 0.87 0.89 0.005
0.79 0.81 0.83 0.81 0.80 0.81 0.008
0.81 0.81 0.004
0.80 0.80 0.005
0.88 0.88 0.003
0.80b 0.81b 0.83a 0.005
0.80ab 0.79b 0.81a 0.006
0.4543 0.0009 0.1444
0.8851 0.0105 0.0196
Ala
Tyr
CP
0.76 0.78 0.80 0.79 0.77 0.79 0.008
0.82 0.84 0.85 0.84 0.83 0.84 0.006
0.82b 0.84ab 0.85a 0.84a 0.82b 0.84a
0.007
0.78b 0.80a 0.81a 0.81a 0.80a 0.81a 0.007
0.81 0.81 0.004
0.78 0.78 0.005
0.84 0.84 0.004
0.84 0.83 0.004
0.80 0.81 0.004
0.87b 0.88b 0.89a 0.003
0.80b 0.80b 0.82a 0.005
0.77b 0.77b 0.80a 0.006
0.83b 0.83b 0.85a 0.005
0.83b 0.83b 0.85a 0.005
0.80 0.80 0.81 0.005
0.6908 0.0001 0.0892
0.8127 0.0245 0.0568
0.6295 0.0110 0.1340
0.9430 0.0104 0.1069
0.8956 0.0052 0.0122
0.0612 0.0877 0.0104
within the same column with no common superscript differ significantly (P ≤ 0.05). represent the average response of 8 replicate pens (32 chicks)/treatment.
Gly
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SEM Ca 1.03 0.64 SEM Phytase 0 500 5,000 SEM P-value Ca Phytase Ca × phytase
Apparent ileal digestibility coefficient
LIMESTONE AND PHYTASE
ated with an increase in the Ca requirement of broilers fed a high Ca diet, as indicated by the increase in bone ash of broilers fed 1.03% Ca compared with broilers fed 0.64% Ca. Supplementing a high level of dietary phytase (5,000 FTU/kg) improved AID of AA, and this has been previously reported (Dilger et al., 2004; Cowieson et al., 2006). Dietary phytase at 5,000 FTU/kg improved AID of serine, tyrosine, and total CP and tended to improve AID of arginine, leucine, phenyalanine, glutamine, and proline but only when broilers were fed the higher level of Ca. Chicken pepsin is largely composed of serine, glutamine, leucine, and tyrosine (Bohak, 1969), AA particularly affected by Ca and phytase in this experiment, and pepsin has a pH-activity curve between 1.8 and 5.0 with an optimum at 2.8 (Bohak, 1969). The data may indicate pH has a substantial effect on pepsin activity, which in turn influences the interaction between Ca and phytase on AA digestibility. In addition, the effect of pH on Ca-phytate interactions (Selle et al., 2009), phytate interference with pepsin activity (Liu et al., 2009), and the influence of Ca on phytase efficacy (Tamim and Angel, 2003) may influence protein digestion in the gizzard and subsequent AA digestibility. It is of interest to note that for the Ca × phytase interaction, optimal distal gizzard pH (for pepsin activity) tended to mirror maximum digestibility of serine, tyrosine, leucine, phenyalanine, glutamine, and proline. For example, gizzard pH and apparent ileal AA digestibility were increased in high Ca diets as phytase increased, with gizzard pH (2.6) near the pepsin pH optimum (2.8) at 5,000 FTU/kg of phytase in 1.03% Ca diets. However, when 0.64% Ca was fed, 500 FTU/kg of phytase reduced gizzard pH (2.15) and reduced serine, glutamine, proline, and tyrosine digestibility—AA largely present in pepsin. Feeding phytase at 5,000 FTU/kg in low Ca diets increased gizzard pH (2.5) back to that near the pepsin pH optimum and also improved apparent ileal AA digestibility. In addition to hydrolyzing phytate, high doses of phytase may improve pepsin efficacy through an increase in gastric pH, resulting in a reduction in serine-, proline-, glutamine-, and tyrosinerich endogenous secretions, which may in turn improve AID of those specific AA. It is concluded that feeding low Ca diets did not affect growth performance of broilers from 1 to 16 d of age, but low Ca diets reduced broiler tibia ash. Dietary phytase at doses above recommended levels improved broiler FCR, tibia ash, and AA digestibility, even in low Ca diets. Phytate, phytase, and Ca had a substantial influence on gastrointestinal pH. High doses of phytase hydrolyze phytate, which may be acidogenic, thereby increasing gastric pH closer to the pepsin pH optima, improving pepsin efficacy, reducing endogenous pepsin secretion, and improving apparent ileal AA digestibility. Feeding phytase at doses above industry recommendations may allow for reduced Ca diets while maintaining broiler performance, bone ash, and improving AA digestibility.
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ciated with dietary treatments, especially when experiments use a small number of animals or replicate pens. In the current experiment, dietary inclusion of 5,000 FTU/kg of phytase influenced gastrointestinal pH. Supplementation of a microbial phytase at 250, 500, or 1,000 FTU/kg did not influence stomach pH in 32kg pigs (Radcliffe et al., 1998), gastrointestinal pH in 42-d-old broilers (Nourmohammadi et al., 2011), or ileal pH in 21-d-old broilers (Akyurek et al., 2011). However, feeding 1,000 FTU/kg of phytase significantly increased fecal pH in 53-kg pigs (Metzler et al., 2008). The increase in gastric pH associated with high levels of supplemental phytase may be directly related to the breakdown of the phytate molecule by phytase. For example, in vitro-simulated gastric pH was significantly reduced from 3.5 to 3.2 (~9%) as synthetic phytate was added to corn-soy diet (C. L. Walk, unpublished data). In addition, by chelating, reducing the digestibility, and increasing the excretion of minerals, such as Na, Ca, P, S, or K (Cowieson et al., 2004; Woyengo et al., 2009), phytate may in fact reduce the dietary electrolyte balance, creating a more acidogenic environment in the gastrointestinal tract. Patience and Chaplin (1997) reported whole venous blood and urinary pH were reduced in 40-kg pigs fed diets with a dietary undetermined anion balance of 175 mEq/kg compared with pigs fed diets with a dietary undetermined anion balance of 454 mEq/kg. In the current experiment, gizzard pH increased approximately 10% as dietary phytase increased from 500 FTU/kg to 5,000 FTU/kg. Therefore, high dietary phytase (5,000 FTU/kg) may have removed enough phytate to ameliorate the acidogenic influence of phytate in the gastrointestinal tract of broilers, which increased gastrointestinal pH. While previous authors have reported improvements in Ca or P digestibility when dietary phytase was supplemented in broiler (Ravindran et al., 2008) or piglet diets (Woyengo et al., 2009), phytase did not influence AID of Ca or P in the current experiment. Sebastian et al. (1996) and Powell et al. (2011) reported an improvement in P digestibility but not Ca digestibility with phytase supplementation in broiler diets. Phytase is more efficacious in P-deficient diets than P-adequate diets (Watson et al., 2006). In the current experiment, dietary P may not have been limiting, as indicated by similar growth performance between the nonsupplemented and phytase-supplemented diets, which led to similar digestibility among all the diets. However, reducing dietary Ca may have improved P digestibility through the reduction of the formation of insoluble Caphosphate complexes (De Kort et al., 2009). In the current study, increasing dietary Ca significantly improved Ca digestibility, and this is counter to the findings of Sebastian et al. (1996). However, Powell et al. (2011) reported no influence of dietary Ca on AID of Ca in broilers and Liesegang et al. (2007) reported increasing dietary Ca from 0.64 to 1.29% increased Ca digestibility in Hermann’s tortoises. The increase in Ca digestibility as Ca increased in the diets may be associ-
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REFERENCES
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Akyurek, H., M. L. Ozduven, A. A. Okur, F. Koc, and H. E. Samli. 2011. The effects of supplementing an organic acid blend and/ or microbial phytase to a corn-soybean-based diet fed to broiler chickens. Afr. J. Agric. Res. 6:642–649. AOAC. 1970. Animal Feeds. Phosphorus 7.095–7.098. Photometric method. Page 125 in Official Methods of Analysis. Assoc. Off. Anal. Chem., Washington, DC. AOAC. 2006. Official Methods of Analysis. 18th ed. Assoc. Off. Anal. Chem., Washington, DC. Augspurger, N. R., and D. H. Baker. 2004a. High dietary phytase levels maximize phytate-phosphorus utilization but does not affect protein utilization in chicks fed phosphorus- or amino aciddeficient diets. J. Anim. Sci. 82:1100–1107. Augspurger, N. R., and D. H. Baker. 2004b. Phytase improves dietary calcium utilization in chicks, and oyster shell, carbonate, citrate, and citrate-malate forms of calcium are equally bioavailable. Nutr. Res. 24:293–301. Ballam, G. C., T. S. Nelson, and L. K. Kirby. 1984. Effect of fiber and phytate source and of calcium and phosphorus level on phytate hydrolysis in the chick. Poult. Sci. 63:333–338. Bohak, Z. 1969. Purification and characterization of chicken pepsinogen and chicken pepsin. J. Biol. Chem. 244:4638–4648. Cowieson, A. J., T. Acamovic, and M. R. Bedford. 2004. The effects of phytase and phytic acid on the loss of endogenous amino acids and minerals from broiler chickens. Br. Poult. Sci. 45:101–108. Cowieson, A. J., T. Acamovic, and M. R. Bedford. 2006. Supplementation of corn-soy-based diets with an Escherichia coli-derived phytase: Effects on broiler chick performance and the digestibility of amino acids and metabolizability of minerals and energy. Poult. Sci. 85:1389–1397. Cowieson, A. J., and V. Ravindran. 2007. Effect of phytic acid and microbial phytase on the flow and amino acid composition of endogenous protein at the terminal ileum of growing broiler chickens. Br. J. Nutr. 98:745–752. De Kort, E., M. Minor, T. Snoeren, T. Van Hooijdonk, and E. Van Der Linden. 2009. Calcium-binding capacity of organic and inorganic ortho- and polyphosphates. Dairy Sci. Technol. 89:283– 299. 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. Engelen, A. J., F. C. van der Heeft, P. H. G. Randsdorp, and W. A. C. Somers. 2001. Determination of phytase activity in feed by colorimetric enzymatic method: Collaborative interlaboratory study. J. AOAC Int. 84:629–633. Gordon, R. W., and D. A. Roland. 1997. The influence of environmental temperature on in vivo limestone solubilization, feed passage rate, and gastrointestinal pH in laying hens. Poult. Sci. 76:683–688. Guinotte, F., J. Gautron, Y. Nys, and A. Soumarmon. 1995. Calcium solubilization and retention in the gastrointestinal tract in chicks (Gallus domesticus) as a function of gastric acid secretion inhibition and of calcium carbonate particle size. Br. J. Nutr. 73:125–139. Hurwitz, S., D. Dubrov, U. Eisner, G. Risenfeld, and A. Bar. 1978. Phosphate absorption and excretion in the young turkey, as influenced by calcium intake. J. Nutr. 108:1329–1335. Liesegang, A., J. M. Hatt, and M. Wanner. 2007. Influence of different dietary calcium levels on the digestibility of Ca, Mg, and P in Hermann’s tortoises (Testudo hermanni). J. Anim. Physiol. Anim. Nutr. (Berl.) 91:459–464. Liu, N., Y. J. Ru, F. D. Li, J. P. Wang, and X. Q. Lei. 2009. Effect of dietary phytate and phytase on proteolytic digestion and growth regulation in broilers. Arch. Anim. Nutr. 63:292–303. Metzler, B. U., R. Mosenthin, T. Baumgartel, and M. Rodehutscord. 2008. The effect of dietary phosphorus and calcium level, phytase supplementation, and ileal infusion of pectin on the chemical
composition and carbohydrase activity of fecal bacteria and the level of microbial metabolites in the gastrointestinal tract of pigs. J. Anim. Sci. 86:1544–1555. Nelson, T. S., J. J. McGillivray, T. R. Shieh, R. J. Wodzinski, and J. H. Ware. 1968. Effect of phytate on the calcium requirement of chicks. Poult. Sci. 47:1985–1989. Nourmohammadi, R., S. M. Hosseini, H. Saraee, and A. Arab. 2011. Plasma thyroid hormone concentrations and pH values of some GI-tract segments of broilers fed on different dietary citric acid and microbial phytase levels. Am. J. Anim. Vet. Sci. 6:1–6. Patience, J. F., and R. K. Chaplin. 1997. The relationship among dietary undetermined anion, acid-base balance, and nutrient metabolism in swine. J. Anim. Sci. 75:2445–2452. Perkin Elmer Instruments. 1998. Notes on mineral analysis using flame atomic absorption. Pages 64–65 in Analyst 800 Atomic Absorption Spectrophotometer User’s Guide. Perkin Elmer Instruments, Ueberlingen, Germany. Powell, S., T. D. Bidner, and L. L. Southern. 2011. Phytase supplementation improved growth performance and bone characteristics in broilers fed varying levels of dietary calcium. Poult. Sci. 90:604–608. Qian, H., E. T. Kornegay, and D. M. Denbow. 1997. Utilization of phytate phosphorus and calcium as influenced by microbial phytase, cholecalciferol, and the calcium:total phosphorus ratio in broiler diets. Poult. Sci. 76:37–46. Radcliffe, J. S., Z. Zhang, and E. T. Kornegay. 1998. The effect of microbial phytase, citric acid, and their interaction in a corn-soybean meal-based diet for weanling pigs. J. Anim. Sci. 76:1880– 1886. Ravindran, V., A. J. Cowieson, and P. H. Selle. 2008. Influence of dietary electrolyte balance and microbial phytase on growth performance, nutrient utilization, and excreta quality of broiler chickens. Poult. Sci. 87:677–688. Sebastian, S., S. P. Touchburn, E. R. Chavez, and P. C. Lague. 1996. Efficacy of supplemental microbial phytase at different dietary calcium levels on growth performance and mineral utilization of broiler chicks. Poult. Sci. 75:1516–1523. Selle, P. H., A. J. Cowieson, and V. Ravindran. 2009. Consequences of calcium interactions with phytate and phytase for poultry and pigs. Livest. Sci. 124:126–141. Shafey, T. M., M. W. McDonald, and J. G. Dingle. 1991. Effects of dietary calcium and available phosphorus concentration on digesta pH and on the availability of calcium, iron, magnesium, and zinc from the intestinal content of meat chickens. Br. Poult. Sci. 32:185–194. Shirley, R. B., and H. M. Edwards Jr.. 2003. Graded levels of phytase past industry standards improves broiler performance. Poult. Sci. 82:671–680. Short, F. J., P. Gorton, J. Wiseman, and K. N. Boorman. 1996. Determination of titanium dioxide added as an inert marker in chicken digestibility studies. Anim. Feed Sci. Technol. 59:215– 221. Simpson, C. J., and A. Wise. 1990. Binding of zinc and calcium to inositol phosphates (phytate) in vitro. Br. J. Nutr. 64:225–232. Tamim, N. M., and R. Angel. 2003. Phytate phosphorus hydrolysis as influenced by dietary calcium and micro-mineral source in broiler diets. J. Agric. Food Chem. 51:4687–4693. Tamim, N. M., R. Angel, and M. Christman. 2004. Influence of dietary calcium and phytase on phytate phosphorus hydrolysis in broiler chickens. Poult. Sci. 83:1358–1367. Watson, B. C., J. O. Matthews, L. L. Southern, and J. L. Shelton. 2006. The effects of phytase on growth performance and intestinal transit time of broilers fed nutritionally adequate diets and diets deficient in calcium and phosphorus. Poult. Sci. 85:493–497. Woyengo, T. A., A. J. Cowieson, O. Adeola, and C. M. Nyachoti. 2009. Ileal digestibility and endogenous flow of minerals and amino acids: Responses to dietary phytic acid in piglets. Br. J. Nutr. 102:428–433.
Key words: broiler, calcium, phosphorus, phytase 2012 Poultry Science 91:1371–1378 http://dx.doi.org/10.3382/ps.2011-01928
INTRODUCTION The amount of Ca required to sustain the broilers’ physiological requirement may vary with age, Ca source, and the phytate content of the diet. For example, if a normal corn-soybean meal diet contains 1% phytate, which can chelate 0.36% Ca (Nelson et al., 1968), dietary phytase may liberate up to 0.36% Ca with complete phytate hydrolysis. Shirley and Edwards (2003) reported 12,000 U of phytase/kg of diet hydrolyzed 95% of the phytate phosphorus in corn and soybean meal diets. Thus, complete phytate hydrolysis may allow for significant reductions in dietary Ca without influencing broiler performance. High dietary Ca has been implicated in reduced phytase efficacy (Ballam et al., 1984; Tamim and Angel, 2003; Tamim et al., 2004), reduced animal performance ©2012 Poultry Science Association Inc. Received October 9, 2011. Accepted February 29, 2012. 1 Corresponding author: [email protected]
(Sebastian et al., 1996; Powell et al., 2011), increased gastrointestinal pH (Guinotte et al., 1995), and interference with macromineral absorption (Hurwitz et al., 1978; Simpson and Wise, 1990). Therefore, reducing dietary Ca and removing Ca chelators, such as phytate, by supplementing high levels of dietary phytase may improve the bird’s utilization efficiency of dietary Ca and P. Reductions in dietary Ca may also indirectly improve amino acid (AA) utilization via a reduction in gastric pH and improved pepsin efficacy. Chicken pepsin activity is pH dependent, with an optimum activity at pH 2.8 (Bohak, 1969), and as a consequence, high dietary Ca may result in unfavorable pH in the proventriculus/gizzard. An increase in gizzard pH significantly reduced Ca solubility in broilers (Guinotte et al., 1995), and a higher pH has been implicated in Ca-phytate interactions in the gastrointestinal tract, as reviewed by Selle et al. (2009). Phytase may spare excess pepsin secretion by hydrolyzing phytate and alleviating the negative effect of phytate on gastric protein hydrolysis and resultant AA digestibility (Cowieson and Ravindran, 2007). High dietary Ca may influence
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ABSTRACT An experiment was conducted to determine the influence of 2 levels of dietary Ca from limestone and 3 levels of phytase on broiler performance, bone ash, gastrointestinal pH, and apparent ileal digestibility (AID) of Ca, P, and amino acids. Cobb 500 broilers (n = 576) were allowed access to one of 6 cornsoy diets from 0 to 16 d. Experimental diets contained 1.03% or 0.64% Ca from limestone and 0.61% total P. Each diet was supplemented with 0, 500, or 5,000 FTU/kg of phytase to create a 2 × 3 factorial experiment. Broiler feed intake (FI) and BW gain were not affected by dietary Ca or phytase. Feed conversion ratio was improved (P < 0.05) as dietary phytase increased (1.36, 1.34, and 1.31, respectively). Tibia ash percent was reduced (P < 0.05) from 41.4 to 40.0% as dietary Ca decreased but increased with phytase addition (P < 0.05). Gizzard and ileal pH were reduced (P < 0.05) in broilers fed 0.64% Ca compared with broilers fed 1.03%
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gastrointestinal pH and directly reduce protein digestion through reduced pepsin efficacy or indirectly influence Ca-phytate precipitation. Dietary phytase may hydrolyze phytate, reducing Ca-phytate interactions, and allow for reduced dietary Ca from limestone while maintaining broiler performance. Reduced dietary Ca in the presence of phytase may also spare pepsin efficacy and improve AA digestibility through a reduction in proximal gastric pH. The objectives of this trial were to determine the influence of 3 levels of phytase in low or high Ca diets on broiler performance, gastrointestinal pH, tibia ash, and apparent ileal digestibility (AID) of Ca, P, and AA.
MATERIALS AND METHODS Birds and Husbandry
Dietary Treatments Experimental diets were arranged as a 2 × 3 factorial including 2 levels of Ca (1.03 and 0.64%) and 3 levels of phytase (0, 500, and 5,000 FTU/kg; AB Vista Feed Ingredients, Marlborough, UK). This resulted in a total of 6 treatment groups replicated by 16 pens of 6 chicks each (96 chicks/dietary treatment). Diets were fed in mash form and based on corn and soybean meal to meet or exceed Cobb nutrient requirements according to the Cobb 500 Broiler Performance and Nutrient Supplement (2008; Siloam Springs, Arkansas) with the exception of Ca and available P (Table 1). Corn was added in place of phytase, and titanium dioxide was added as an inert marker for nutrient digestibility evaluation. The phytase used in the experiment was an Escherichia coli 6-phytase expressed in Pichia pastoris with an expected activity of 5,000 FTU/g. 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. All experimental procedures were approved by the Virginia Tech Institutional Animal Care and Use Committee.
Response Variables Birds were weighed by pen before placement (d 0) and on d 16 to measure mean treatment BW and calculate mean BW gain. Feed intake (FI) was also measured on d 0 and d 16 and used to calculate feed conversion (FCR). Mortality was recorded daily, and any
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Cobb 500, male broilers (576) were obtained from a commercial hatchery at day of hatch and transported to the research facilities at Virginia Tech. On arrival, chicks were randomized, weighed by pen, and placed in Petersime battery brooders with respect to 6 dietary treatments. Birds were maintained on a constant lighting program and allowed ad libitum access to the treatment diets for the duration of the trial (d 0 to 16).
birds culled or dead were weighed. Treatment FI and FCR were adjusted according to the number of bird days, which is defined as the number of bird days alive in each cage multiplied by the number of days without incidence of mortality. Birds for sampling were stunned by exposure to CO2 gas for approximately 30 s and euthanized by cervical dislocation, for collection of tibias and digesta. On d 16, 2 birds/pen of average BW were killed to obtain measurements of pH from the proximal gizzard (proventricular opening), distal gizzard (duodenal opening), duodenum (duodenum loop), medial jejunum (defined as the intestinal section distal to the duodenal loop and proximal to the Meckel’s diverticulum), proximal ileum (approximately 5 cm distal to the Meckel’s diverticulum), and the distal ileum (approximately 2 cm proximal to the ileocecal junction). From each bird, pH measurements were obtained directly from the digesta contents in the lumen using a digital pH meter (Mettler-Toledo, Columbus, OH) and spear-tip electrode (Sensorex, Garden Grove, CA). The pH values from each gastrointestinal section/bird were then pooled to obtain a mean pH value/pen. Left tibias were obtained from 3 birds/pen on d 16 and pooled for determination of bone ash. Tibias were stripped of adhering tissues, wrapped in cheesecloth, and dried overnight at 100°C. Fat was extracted from the tibias using a Soxhelt apparatus and 100% ethyl ether according to modified methods of Watson et al. (2006). Fat-extracted tibias were dried for 24 h at 100°C and ashed in a muffle furnace for 24 h at 600°C to determine bone ash. Digesta was collected from the ileum, defined as the Meckel’s diverticulum to the ileocecal junction, from the 5 birds/pen euthanized to measure pH and tibias. The collected digesta was pooled by pen, weighed, and frozen at −20°C until further chemical analysis. Prior to analysis, digesta samples were lyophilized and ground to pass a 1-mm screen. Amino acid concentrations in the diet and ileal digesta were determined by HPLC according to methods described by the AOAC (2006) for standard protein hydrolysis (method 45.3.05). Diets and ileal digesta were subjected to a total Ca and P analysis using a nitric acid and perchloric acid wet ash digestion. Phosphorus in the diets and digesta was analyzed colorimetrically using a Genesys 5 spectrophotometer (Thermo Electron Corp., Madison, WI) at 410 nm according to methods of the AOAC (1970) for P (method 7.095–7.098). Calcium in the diets and digesta was obtained using a Perkin Elmer Atomic Absorption spectrophotometer (Analyst 800, Ueberlingen, Germany) and 1% lanthanum oxide according to Analyst 800 Atomic Absorption Spectrophotometer User’s Guide (Perkin Elmer Insruments, 1998) methods. Phytase recovered in the diets was analyzed by Enzyme Services and Consultancy (Blackwood, Wales) according to modified methods of Engelen et al. (2001). Titanium dioxide in the diet and ileal digesta was analyzed ac-
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LIMESTONE AND PHYTASE Table 1. Composition and nutrient content of experiment diets Item (%)
Low Ca
59.85 29.16 4.00 2.50 1.36 0.77 1.21 0.33 0.16 0.23 0.10 0.10 0.10 0.01 0.01 0.10 87.29 21.00 1.20 0.80 0.89 0.90 0.53 0.32 3,003 21.79 1.36 0.82 1.03 0.61
61.51 28.88 4.00 2.50 0.77 0.77 0.43 0.33 0.17 0.23 0.10 0.10 0.10 0.01 0.01 0.10 87.10 21.00 1.20 0.80 0.89 0.60 0.53 0.32 3,003 22.39 1.37 0.82 0.64 0.61
1Dicalcium
phosphate supplied 18.5% P and 22% Ca. supplied 38% Ca. 3Supplied per kilogram of diet: iron (ferrous sulfate), 40 mg; manganese (manganese sulfate and manganous oxide), 120 mg; zinc (zinc oxide), 210 mg; cobalt (cobalt carbonate), 2.2 mg; iodine (calcium iodate), 132 mg. 4Supplied per kilogram of diet: vitamin A, 8,818 IU; vitamin D , 2,646 ICU; vitamin E, 22 IU; vitamin B , 26 3 12 μg; riboflavin, 8.8 mg; niacin, 88 mg; d-pantothenic acid, 22 mg; vitamin K, 2.6 mg; folic acid, 2.2 mg; vitamin B6, 4.3 mg; thiamine, 3.7 mg; d-biotin, 220 μg. 5Supplied per kilogram of diet: 600 ppm. 6Corn was supplemented in place of phytase in diets containing 0 or 500 FTU/kg of phytase to equal 100%. 2Limestone
cording to methods of Short et al. (1996). Diet and ileal titanium dioxide, AA, Ca, and P values were used to calculate AID using the following equation: Digestibility coefficient = [(nutrient/TiO2)diet – (nutrient/TiO2)ileum]/(nutrient/TiO2)diet.
Statistical Analysis Data was analyzed as a 2 × 3 factorial using the fit model platform of JMP 8.0 (SAS Institue Inc., Cary, NC). Pen served as the experimental unit for all parameters measured. The statistical model included Ca, phytase, and the interaction of Ca × phytase. When differences were significant, means were separated using Tukey’s honestly significant difference test. Significance was accepted at P ≤ 0.05. In the event that interactions were not significant, main effects were discussed.
RESULTS The analyzed dietary Ca and P were within acceptable ranges and in agreement with formulated values when mixing and assay errors were considered (Table 1). Phytase activities recovered in the diets were approximately 37% higher than formulated, and these results may be associated with an actual phytase activity higher than expected, sample variation and mixing, and assay errors. Feed intake and BW gain were not influenced by dietary Ca, phytase, or the interaction of Ca × phytase (Table 2). Phytase supplementation at 500 FTU/kg improved (P ≤ 0.05) FCR compared with broilers fed 0 FTU/kg, and supplementation at 5,000 FTU/kg improved (P ≤ 0.05) FCR compared with broilers fed 0 or 500 FTU/kg (1.36, 1.34, and 1.31, respectively; Table 2). Feed conversion was not influenced by dietary Ca or the interaction of Ca × phytase. Overall, experimental mortality was low (3.41%), but
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Ingredient Corn Soybean meal, 45% Dried distillers grains with solubles, 26% Poultry by-product, 58% Poultry fat Dicalcium phosphate1 Limestone2 Salt l-Lysine HCl dl-Methionine Titanium dioxide Trace mineral premix3 Vitamin premix4 l-Threonine Selenium premix5 Phytase6 Calculated composition DM CP Lysine Threonine Total sulfur amino acids Ca Total P Available P Nutrient composition Energy (ME; kcal/kg) Analyzed composition CP Lysine Threonine Ca Total P
High Ca
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Table 2. Influence of Ca and phytase on growth performance, mortality, and tibia ash of broilers1 from d 0 to 16 Dietary treatment Ca (%)
Phytase (FTU)
1.03 0.64
BW gain (g)
FCR2
Mortality (%)
Tibia ash (%)
626.9 629.2 621.1 634.1 623.6 616.6 7.679 625.7 624.8 4.433 630.5 626.4 618.8 5.430 0.8778 0.3100 0.6565
462.3 474.8 476.3 468.3 463.3 472.1 5.627 471.1 467.9 3.249 465.3 469.1 474.2 3.979 0.4843 0.2890 0.3053
1.356 1.327 1.305 1.354 1.346 1.306 0.0085 1.329 1.335 0.0049 1.355a 1.337b 1.306c 0.0060 0.3877 0.0001 0.3938
1.04 2.08 4.17 0.00 4.17 8.33 2.43 4.17 0.52b 3.13ab 6.25a 0.2590 0.0059 0.3664
40.07 41.50 42.72 39.19 39.93 40.80 0.2455 41.43a 39.97b 0.1417 39.63c 40.71b 41.76a 0.1736 0.0001 0.0001 0.1029
a–cMeans
within the same column with no common superscript differ significantly (P ≤ 0.05). represent the average response of 16 replicate pens (96 chicks)/treatment. 2FCR = feed conversion ratio, feed:gain; corrected for mortality. 1Means
broilers fed 5,000 FTU/kg of phytase had an increase (P ≤ 0.05) in percent mortality compared with broilers fed 0 FTU/kg (6.25 vs. 0.52%, respectively; Table 2). Tibia ash was not influenced by the interaction of Ca × phytase (Table 2). Reducing dietary Ca from 1.03% to 0.64% reduced (P ≤ 0.05) broiler tibia ash (41.4 and 40.0%, respectively). Phytase supplementation at 500 FTU/kg increased (P ≤ 0.05) tibia ash compared with broilers fed 0 FTU/kg phytase. Broilers fed 5,000 FTU/kg phytase had increased (P ≤ 0.05) tibia ash compared with broilers fed 500 FTU/kg. Gastrointestinal pH was not influenced by Ca × phytase interaction (Table 3). Distal gizzard and distal ileal pH was lower (P ≤ 0.05) in broilers fed 0.64% Ca compared with broilers fed 1.03% Ca. Supplementing broiler diets with 5,000 FTU/kg of phytase increased (P ≤ 0.05) pH in the gastrointestinal tract compared with broilers fed 0 or 500 FTU/kg of phytase, except in the distal gizzard where 5,000 FTU/kg resulted in higher pH compared with 500 FTU/kg of supplementation, but neither of these were different from broilers fed 0 FTU/kg of phytase. Apparent ileal P or Ca digestibility was not influenced by a Ca × phytase interaction or by phytase supplementation (Table 4). Reducing dietary Ca from 1.03% to 0.64% improved (P ≤ 0.05) AID of P approximately 7%. Apparent ileal Ca digestibility was reduced (P ≤ 0.05) approximately 21% in broilers fed 0.64% Ca compared with broilers fed 1.03% Ca. Apparent ileal serine, tyrosine, and total CP digestibility were influenced (P ≤ 0.05) by a Ca × phytase interaction (Tables 5 and 6). In general, increasing phytase improved AID of serine, tyrosine, or total CP, when broilers were fed 1.03% Ca. However, when broil-
ers were fed reduced (0.64%) Ca diets, phytase supplementation did not influence or negatively influenced AID of serine, tyrosine, or total CP, which resulted in a Ca × phytase interaction. There were no other Ca × phytase interactions influencing AID of AA. However, the AID of all AA was influenced by the main effect of phytase, with the exception of threonine or histidine (Tables 5 and 6). In general, phytase supplementation at 5,000 FTU/kg improved (P ≤ 0.05) the AID of AA approximately 2 to 4% compared with phytase at 0 or 500 FTU/kg. The main effect of Ca did not influence AID of AA.
DISCUSSION Broiler FI or BW gain was not influenced by dietary Ca or phytase, which would suggest Ca or P were not limiting growth performance. Sebastian et al. (1996) reported no differences in FI or BW gain when 21-d-old broilers were fed corn-soy diets formulated to contain 0.60% or 1.0% Ca. However, Augspurger and Baker (2004b) observed a linear improvement in BW gain as dietary Ca increased from 0 to 1.0%. In addition, phytase supplementation improved BW gain in low Ca diets (Sebastian et al., 1996; Augspurger and Baker, 2004b; Powell et al., 2011), suggesting phytase may improve Ca utilization by hydrolyzing phytate. Although the overall mortality in the experiment was low (3.41%), the increase in mortality associated with 5,000 FTU/kg of phytase was not expected. The use of a small population of birds in the experiment, necropsy of the dead birds, and growth performance and bone ash of the remaining birds would indicate mortality was not the result of any negative biological function of
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SEM Ca 1.03 0.64 SEM Phytase 0 500 5,000 SEM P-value Ca Phytase Ca × phytase
0 500 5,000 0 500 5,000
Feed intake (g)
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LIMESTONE AND PHYTASE Table 3. Influence of Ca and phytase on
broiler1
Dietary treatment Ca (%)
Gizzard
Duodenum
Jejunum
Ileum
Proximal
Distal
Loop
Medial
Proximal
Distal
0 500 5,000 0 500 5,000
1.94 1.84 2.18 1.85 1.76 2.09 0.075 1.98 1.90 0.043 1.89b 1.80b 2.13a 0.053 0.1707 0.0001 0.9921
2.40 2.54 2.63 2.44 2.15 2.53 0.090 2.52a 2.37b 0.052 2.42ab 2.34b 2.58a 0.064 0.0409 0.0330 0.0574
6.22 6.18 6.26 6.14 6.17 6.26 0.029 6.22 6.19 0.017 6.18b 6.17b 6.26a 0.020 0.1672 0.0066 0.3541
5.90 5.96 6.01 5.91 5.86 6.00 0.028 5.95 5.92 0.016 5.90b 5.91b 6.00a 0.019 0.1831 0.0008 0.1189
5.86 5.90 5.97 5.87 5.85 5.91 0.030 5.91 5.88 0.017 5.86b 5.88b 5.94a 0.021 0.1723 0.0276 0.4356
6.63 6.74 7.06 6.49 6.55 6.92 0.078 6.81a 6.65b 0.045 6.56b 6.65b 6.99a 0.055 0.0165 0.0001 0.9255
0.64
a,bMeans 1Means
within the same column with no common superscript differ significantly (P ≤ 0.05). represent the average response of 16 replicate pens (32 chicks)/treatment.
phytase but rather the loss of 2 birds in one pen that equaled 33% mortality for that particular replicate. Increasing dietary phytase significantly improved broiler FCR and bone ash, and this has been previously reported in broilers fed diets containing supplemental phytase in excess of 1,000 FTU/kg (Shirley and Edwards, 2003; Augspurger and Baker, 2004a). Reducing dietary Ca significantly reduced broiler tibia ash, even though broiler growth performance was not influenced by Ca level, which would suggest that Ca was limiting for this parameter and that tibia ash is a more
sensitive indicator of mineral status than growth performance. Percentage of tibia ash tended to decrease in broilers fed 0.60% Ca compared with broilers fed 1.0% Ca (Sebastian et al., 1996). However, Powell et al. (2011) reported an improvement in percentage of tibia ash as dietary Ca was reduced from 1.33% to 0.67%. The differences in the published literature may be related to the level of P in the experimental diets and the Ca:P ratio. For example, Powell et al. (2011) fed 0.20% nonphytate phosphorus, whereas Sebastian et al. (1996) fed 0.31% available P, which was closer to the
Table 4. Influence of Ca and phytase on apparent ileal Ca and P digestibility of 16-d-old broilers1 Apparent ileal digestibility (digestibility coefficient)
Dietary treatment Ca (%) 1.03 0.64 SEM Ca 1.03 0.64 SEM Phytase 0 500 5,000 SEM P-value Ca Phytase Ca × phytase a,bMeans 1Means
Phytase (FTU)
P
Ca
0 500 5,000 0 500 5,000
0.69 0.68 0.67 0.75 0.71 0.73 0.0299 0.68b 0.73a 0.0173 0.72 0.69 0.70 0.0217 0.0410 0.6933 0.9375
0.66 0.67 0.69 0.52 0.56 0.51 0.0454 0.67a 0.53b 0.0264 0.59 0.61 0.60 0.0322 0.0004 0.8447 0.7583
within the same column with no common superscript differ significantly (P ≤ 0.05). represent the average response of 16 replicate pens (64 chicks)/treatment.
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Phytase (FTU)
1.03
SEM Ca 1.03 0.64 SEM Phytase 0 500 5,000 SEM P-value Ca Phytase Ca × phytase
gastrointestinal pH from d 0 to 16
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Table 5. Influence of Ca and phytase on apparent ileal essential amino acid digestibility of 16-d-old broilers1 Dietary treatment Phytase (FTU)
Ca (%) 1.03
0 500 5,000 0 500 5,000
0.64
Thr
Arg
Val
Iso
Leu
Phe
Lys
His
0.75 0.76 0.78 0.77 0.75 0.77 0.009 0.76 0.76 0.005 0.76 0.76 0.77 0.007 0.7753 0.2072 0.1360
0.87 0.89 0.90 0.89 0.88 0.90 0.004 0.89 0.89 0.003 0.88b 0.88b 0.90a 0.003 0.3967 0.0001 0.0574
0.79 0.81 0.83 0.80 0.80 0.82 0.008 0.81 0.81 0.004 0.79b 0.81b 0.82a 0.005 0.8670 0.0028 0.2248
0.81 0.83 0.85 0.83 0.83 0.85 0.007 0.83 0.84 0.004 0.82b 0.83b 0.85a 0.005 0.6737 0.0003 0.2848
0.83 0.85 0.86 0.84 0.84 0.85 0.006 0.85 0.84 0.004 0.84b 0.84b 0.86a 0.004 0.9045 0.0032 0.0790
0.83 0.85 0.87 0.84 0.84 0.86 0.006 0.85 0.85 0.004 0.84b 0.84b 0.86a 0.004 0.8964 0.0001 0.0980
0.86 0.87 0.89 0.88 0.88 0.89 0.005 0.87 0.88 0.003 0.87b 0.87b 0.89a 0.004 0.1232 0.0010 0.2166
0.84 0.85 0.86 0.85 0.85 0.85 0.006 0.85 0.85 0.003 0.84 0.85 0.86 0.004 0.9865 0.1317 0.1336
a,bMeans 1Means
within the same column with no common superscript differ significantly (P ≤ 0.05). represent the average response of 8 replicate pens (32 chicks)/treatment.
level of available P fed in the current experiment. When evaluating the influence of Ca:P ratio on broiler weight gain or toe ash, Qian et al. (1997) reported a 24% or 15% improvement, respectively, in broilers fed low Ca diets with low P (1.1:1, Ca:P ratio) compared with broilers fed high Ca diets with low P (2.0:1 Ca:P ratio). Gizzard and ileal pH were influenced by Ca, indicating high dietary limestone may act as an antacid in the distal portions of the gizzard and ileum. Increasing dietary limestone increased gizzard pH of immature pullets (Guinotte et al., 1995) and increased crop and ileal
pH in 12-d-old broilers (Shafey et al., 1991), but it did not influence gizzard or small intestinal pH in mature laying hens (Guinotte et al., 1995; Gordon and Roland, 1997). Conflicting reports regarding differences in gastrointestinal pH and limestone supplementation may arise from the large variation in gastrointestinal pH between broilers. For example, in the current experiment, the pH in the gizzard or ileum of broilers ranged from approximately 1.0 to 4.5 and 4.5 to 7.5, respectively. Large variation in pH of the specific digestive organs of broilers may confound or mask differences in pH asso-
Table 6. Influence of Ca and phytase on apparent ileal nonessential amino acid digestibility of 16-d-old broilers1 Dietary treatment Ca (%)
Phytase (FTU)
1.03 0.64 SEM Ca 1.03 0.64 SEM Phytase 0 500 5,000 SEM P-value Ca Phytase Ca × phytase a–cMeans 1Means
0 500 5,000 0 500 5,000
Apparent ileal digestibility coefficient Asp
Ser
0.79 0.81 0.83 0.81 0.80 0.83 0.007
0.79bc 0.80abc 0.81a 0.81a 0.78c 0.81ab
Glu
Pro
0.008
0.86 0.88 0.90 0.88 0.87 0.89 0.005
0.79 0.81 0.83 0.81 0.80 0.81 0.008
0.81 0.81 0.004
0.80 0.80 0.005
0.88 0.88 0.003
0.80b 0.81b 0.83a 0.005
0.80ab 0.79b 0.81a 0.006
0.4543 0.0009 0.1444
0.8851 0.0105 0.0196
Ala
Tyr
CP
0.76 0.78 0.80 0.79 0.77 0.79 0.008
0.82 0.84 0.85 0.84 0.83 0.84 0.006
0.82b 0.84ab 0.85a 0.84a 0.82b 0.84a
0.007
0.78b 0.80a 0.81a 0.81a 0.80a 0.81a 0.007
0.81 0.81 0.004
0.78 0.78 0.005
0.84 0.84 0.004
0.84 0.83 0.004
0.80 0.81 0.004
0.87b 0.88b 0.89a 0.003
0.80b 0.80b 0.82a 0.005
0.77b 0.77b 0.80a 0.006
0.83b 0.83b 0.85a 0.005
0.83b 0.83b 0.85a 0.005
0.80 0.80 0.81 0.005
0.6908 0.0001 0.0892
0.8127 0.0245 0.0568
0.6295 0.0110 0.1340
0.9430 0.0104 0.1069
0.8956 0.0052 0.0122
0.0612 0.0877 0.0104
within the same column with no common superscript differ significantly (P ≤ 0.05). represent the average response of 8 replicate pens (32 chicks)/treatment.
Gly
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SEM Ca 1.03 0.64 SEM Phytase 0 500 5,000 SEM P-value Ca Phytase Ca × phytase
Apparent ileal digestibility coefficient
LIMESTONE AND PHYTASE
ated with an increase in the Ca requirement of broilers fed a high Ca diet, as indicated by the increase in bone ash of broilers fed 1.03% Ca compared with broilers fed 0.64% Ca. Supplementing a high level of dietary phytase (5,000 FTU/kg) improved AID of AA, and this has been previously reported (Dilger et al., 2004; Cowieson et al., 2006). Dietary phytase at 5,000 FTU/kg improved AID of serine, tyrosine, and total CP and tended to improve AID of arginine, leucine, phenyalanine, glutamine, and proline but only when broilers were fed the higher level of Ca. Chicken pepsin is largely composed of serine, glutamine, leucine, and tyrosine (Bohak, 1969), AA particularly affected by Ca and phytase in this experiment, and pepsin has a pH-activity curve between 1.8 and 5.0 with an optimum at 2.8 (Bohak, 1969). The data may indicate pH has a substantial effect on pepsin activity, which in turn influences the interaction between Ca and phytase on AA digestibility. In addition, the effect of pH on Ca-phytate interactions (Selle et al., 2009), phytate interference with pepsin activity (Liu et al., 2009), and the influence of Ca on phytase efficacy (Tamim and Angel, 2003) may influence protein digestion in the gizzard and subsequent AA digestibility. It is of interest to note that for the Ca × phytase interaction, optimal distal gizzard pH (for pepsin activity) tended to mirror maximum digestibility of serine, tyrosine, leucine, phenyalanine, glutamine, and proline. For example, gizzard pH and apparent ileal AA digestibility were increased in high Ca diets as phytase increased, with gizzard pH (2.6) near the pepsin pH optimum (2.8) at 5,000 FTU/kg of phytase in 1.03% Ca diets. However, when 0.64% Ca was fed, 500 FTU/kg of phytase reduced gizzard pH (2.15) and reduced serine, glutamine, proline, and tyrosine digestibility—AA largely present in pepsin. Feeding phytase at 5,000 FTU/kg in low Ca diets increased gizzard pH (2.5) back to that near the pepsin pH optimum and also improved apparent ileal AA digestibility. In addition to hydrolyzing phytate, high doses of phytase may improve pepsin efficacy through an increase in gastric pH, resulting in a reduction in serine-, proline-, glutamine-, and tyrosinerich endogenous secretions, which may in turn improve AID of those specific AA. It is concluded that feeding low Ca diets did not affect growth performance of broilers from 1 to 16 d of age, but low Ca diets reduced broiler tibia ash. Dietary phytase at doses above recommended levels improved broiler FCR, tibia ash, and AA digestibility, even in low Ca diets. Phytate, phytase, and Ca had a substantial influence on gastrointestinal pH. High doses of phytase hydrolyze phytate, which may be acidogenic, thereby increasing gastric pH closer to the pepsin pH optima, improving pepsin efficacy, reducing endogenous pepsin secretion, and improving apparent ileal AA digestibility. Feeding phytase at doses above industry recommendations may allow for reduced Ca diets while maintaining broiler performance, bone ash, and improving AA digestibility.
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ciated with dietary treatments, especially when experiments use a small number of animals or replicate pens. In the current experiment, dietary inclusion of 5,000 FTU/kg of phytase influenced gastrointestinal pH. Supplementation of a microbial phytase at 250, 500, or 1,000 FTU/kg did not influence stomach pH in 32kg pigs (Radcliffe et al., 1998), gastrointestinal pH in 42-d-old broilers (Nourmohammadi et al., 2011), or ileal pH in 21-d-old broilers (Akyurek et al., 2011). However, feeding 1,000 FTU/kg of phytase significantly increased fecal pH in 53-kg pigs (Metzler et al., 2008). The increase in gastric pH associated with high levels of supplemental phytase may be directly related to the breakdown of the phytate molecule by phytase. For example, in vitro-simulated gastric pH was significantly reduced from 3.5 to 3.2 (~9%) as synthetic phytate was added to corn-soy diet (C. L. Walk, unpublished data). In addition, by chelating, reducing the digestibility, and increasing the excretion of minerals, such as Na, Ca, P, S, or K (Cowieson et al., 2004; Woyengo et al., 2009), phytate may in fact reduce the dietary electrolyte balance, creating a more acidogenic environment in the gastrointestinal tract. Patience and Chaplin (1997) reported whole venous blood and urinary pH were reduced in 40-kg pigs fed diets with a dietary undetermined anion balance of 175 mEq/kg compared with pigs fed diets with a dietary undetermined anion balance of 454 mEq/kg. In the current experiment, gizzard pH increased approximately 10% as dietary phytase increased from 500 FTU/kg to 5,000 FTU/kg. Therefore, high dietary phytase (5,000 FTU/kg) may have removed enough phytate to ameliorate the acidogenic influence of phytate in the gastrointestinal tract of broilers, which increased gastrointestinal pH. While previous authors have reported improvements in Ca or P digestibility when dietary phytase was supplemented in broiler (Ravindran et al., 2008) or piglet diets (Woyengo et al., 2009), phytase did not influence AID of Ca or P in the current experiment. Sebastian et al. (1996) and Powell et al. (2011) reported an improvement in P digestibility but not Ca digestibility with phytase supplementation in broiler diets. Phytase is more efficacious in P-deficient diets than P-adequate diets (Watson et al., 2006). In the current experiment, dietary P may not have been limiting, as indicated by similar growth performance between the nonsupplemented and phytase-supplemented diets, which led to similar digestibility among all the diets. However, reducing dietary Ca may have improved P digestibility through the reduction of the formation of insoluble Caphosphate complexes (De Kort et al., 2009). In the current study, increasing dietary Ca significantly improved Ca digestibility, and this is counter to the findings of Sebastian et al. (1996). However, Powell et al. (2011) reported no influence of dietary Ca on AID of Ca in broilers and Liesegang et al. (2007) reported increasing dietary Ca from 0.64 to 1.29% increased Ca digestibility in Hermann’s tortoises. The increase in Ca digestibility as Ca increased in the diets may be associ-
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