Efficacy of Phytases on Egg Production and Nutrient Digestibility in Layers Fed Reduced Phosphorus Diets N. Liu,* G. H. Liu,† F. D. Li,*1 J. S. Sands,‡ S. Zhang,† A. J. Zheng,† and Y. J. Ru§ *Faculty of Animal Science and Technology, Gansu Agricultural University, Lanzhou, China, 730070; †Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China, 100081; ‡Danisco Animal Nutrition, Marlborough, Wiltshire, SN8 1XN, United Kingdom; and §Danisco Animal Nutrition, Science Park III, Singapore, 117525 ABSTRACT The effects of phytases on the performance of layers and the ileal nutrient digestibility of corn-, soybean-, and by-product meal-based diets were assessed with 320 Hy-Line brown layers from 23 to 28 wk of age. Layers were grouped randomly into 5 treatments, with 8 replicates per treatment and 8 layers per replicate. The 5 diets consisted of a positive control diet with adequate Ca (3.30%), total P (0.50%), and nonphytate P (NPP; 0.28%), and a negative control diet with Ca reduced by 0.12%, total P reduced by 0.14%, NPP reduced by 0.13%, and 3 phytases (phytase A derived from Aspergillus niger, and phytases B and C derived from Escherichia coli) supplemented at 300 phytase units/kg of feed, respectively. Egg production and feed intake were recorded daily, and eggshell quality and ileal nutrient digestibility were measured at the end of a 6-wk feeding period. The results
revealed that the reduction of Ca and P from the positive control diet significantly depressed feed intake, egg mass, eggshell hardness, and the digestibility of N, Ca, P, and amino acids (P < 0.05). Phytase supplementation in the negative control diet improved the digestibility of P and Ca by 11.08 and 9.81% (P < 0.05), respectively, whereas it improved the digestibility of amino acids by 2 to 8% (P < 0.05). However, the digestibility of most amino acids was not restored to the levels of the positive control diet by the application of phytases. Supplementing phytases in the negative control diet improved the rate of lay, egg mass, and egshell quality to the levels of birds fed the positive control diet. These results suggest that supplementing phytases can improve the digestibility not only of Ca and P, but also of amino acids in layers fed a corn-, soybean-, and by-product-based diet.
Key words: phytase, nutrient, digestibility, layer 2007 Poultry Science 86:2337–2342 doi:10.3382/ps.2007-00079
INTRODUCTION Phytate exists widely in feed ingredients and is poorly degradable in the gastrointestinal tract of poultry because of the lack of endogenous phytase. Its presence in poultry feed ingredients restricts the effective use of organic P and other nutrients, including Ca, energy, and amino acids, in the alimentary tract (Cheryan, 1980; Ravindran et al., 1995). This is due to the chelation of Ca (Vohra et al., 1965; Oberleas, 1973; Cheryan, 1980), amino acids (De Rham and Jost, 1979), and starch (Ravindran et al., 1999) by phytate. This antinutritional effect was demonstrated in broilers, in which the digestibility of energy and amino acids declined with an increase in dietary phytate (Ravindran et al., 2006). Although it has been well documented that phytase hydrolyzes phytate and increases the digestion of P, consequently reducing the excretion of P and
©2007 Poultry Science Association Inc. Received February 15, 2007. Accepted August 4, 2007. 1 Corresponding author:
[email protected]
lowering environment pollution, the use of phytase as a tool to improve amino acid and energy utilization has not been sufficiently demonstrated. Recently, several studies have demonstrated that the application of phytase to conventional diets improves the digestion of energy and amino acids in broilers (Rutherfurd et al., 2004; Onyango et al., 2005; Cowieson et al., 2006b). Based on a large amount of data, the matrix value of phytase has been developed to optimize profits in the broiler production system (Shelton et al., 2004; Partridge, 2006; Selle et al., 2006). The efficacy of phytase on performance and Ca and P digestibility in layers fed a corn- and soybean-based diet has been well established by Van der Klis et al. (1997), Lim et al. (2003), Panda et al. (2005), and Wu et al. (2006). However, only 2 studies have reported the effect of phytase supplementation on the digestibility of amino acids. Jalal et al. (1999) reported that supplementation of phytase in corn and soybean meal diets for layers resulted in significant improvements in the digestibility of Met, Cys, Ala, and Glu. In contrast, Snow et al. (2003) studied the effects of phytase on amino acid digestibility in molted
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laying hens from the age of 78 to 81 wk and found that phytase supplementation of diets based on corn, soybean meal, and meat and bone meal, with 0.25% dietary nonphytate P (NPP) and 3.55% Ca, did not affect hen performance and amino acid digestibility, although an interaction between feed type and phytase on the digestibility of amino acids was detected. Because there is limited information on the effect of phytase on the digestion and utilization of nutrients, especially energy and amino acids, the application of phytase in layer feed has not been optimized according to the diet type and phytate level in the diets. Given the diversity of feed ingredients in layer diets in the Asia-Pacific region, improvements in nutrient digestion similar to those seen in broilers are expected in layers, especially when a large proportion of by-products are included in the layer diet formulation. The current study examined the effect of phytase on feed intake, egg production, eggshell quality, and the digestion of P, Ca, energy, and amino acids in layers fed corn-, soybean-, and by-product-based diets.
MATERIALS AND METHODS Treatments The experiment included 5 dietary treatments. The positive control diet was similar to the commercial diet specifications used in China, but the nutrient specification was lower than that recommended by the NRC (1994). Negative control diets were formulated from the positive control diet, with Ca, P, and NPP levels reduced by 0.12, 0.14, and 0.13%, respectively. The other 3 diets were the negative control diet supplemented with phytase A (derived from Aspergillus niger) or phytases B and C (both derived from Escherichia coli), each at 300 phytase units/ kg of feed. The reductions of Ca and P in the negative control diet and the dose rates of phytases were recommended by the phytase suppliers and are applied by feed producers commercially. The diet formulation is listed in Table 1.
Bird Management A total of 320 Hy-Line brown layers at 23 wk of age were allocated randomly into 5 treatments, with 8 replicates per treatment and 8 layers per replicate. All birds were housed in 3-layered cages and offered feed and water ad libitum. During the experimental period, birds received 16 h/d of manipulated lighting and ventilation at a natural ambient temperature of 30 to 37°C. Egg production and the health status of layers were observed as the adjustment period for 1 wk before the experiment commenced. The experimental diets were fed for 6 wk. Experimental birds were handled with care to avoid unnecessary discomfort, and all experiments were approved by the University of Gansu Agricultural University Animal Care and Use Committee. Feed intake, laying rate, and egg weight were recorded daily for each replicate. In the last week of the trial, all eggs were collected over
Table 1. Composition of the basal diets
Item Ingredient Corn Soybean oil Soybean meal Rapeseed meal Cottonseed meal Corn gluten meal DDGS1 (corn) Lys Met Limestone Dicalcium phosphate Salt Vitamin-mineral premix2 Phytase (A, B, or C3; phytase units/kg) Calculated nutrient level ME [Mcal/kg (MJ/kg)] CP Ca Total P Nonphytate P Lys Met Met + Cys Determined nutrient level Lys Met Ca Total P
Positive control (%) 57.66 1.14 7.47 6.16 8.00 4.00 5.00 0.00 0.08 8.39 0.81 0.30 1.00 0 2.65 (11.08) 16.50 3.30 0.50 0.28 0.58 0.36 0.67 0.66 0.35 3.29 0.50
Negative control (%) 59.49 0.44 7.09 5.00 8.00 5.00 5.00 0.02 0.07 8.59 0.01 0.30 1.00 300 2.65 (11.08) 16.50 3.18 0.36 0.15 0.58 0.36 0.67 0.63 0.34 3.20 0.34
1
DDGS = distillers dried grains with solubles. Provides (per kg of diet): vitamin A (retinyl acetate), 8,000 IU; cholecalciferol, 1,600 IU; vitamin E (DL-α-tocopheryl acetate), 5 IU; vitamin K, 0.5 mg; riboflavin, 2.5 mg; D-pantothenic acid, 2.2 mg; niacin, 20 mg; pyridoxine, 3.0 mg; biotin, 0.10 mg; folic acid, 0.25 mg; vitamin B12, 0.004 mg; choline, 500 mg; manganese, 60 mg; iodine, 0.35 mg; iron, 60 mg; copper, 8 mg; zinc, 80 mg; and selenium, 0.30 mg. 3 A, B, and C are phytases. A is derived from Aspergillus niger; B and C are derived from Escherichia coli. 2
3 d for each replicate, and then 20 eggs from each replicate were selected randomly to measure eggshell quality. At the end of the 6-wk experiment, all diets were removed from the feeders and replaced with the respective diets containing the indigestible marker SiO2. Three days later, 6 birds in each replicate were euthanized and a section of ileum was removed by cutting from the ileo-cecal junction to Meckel’s diverticulum. A 50-mL syringe full of room-temperature deionized water was inserted into one end of the ileum and the digesta was carefully flushed out of the gut into a 10-cm-diameter Petri dish. The digesta from each replicate was pooled and freeze-dried for laboratory analysis.
Chemical Analysis The ileal digestibility coefficients of nutrients were determined by using the inert indicator method (SiO2). The concentrations of CP and P in samples were determined according to AOAC (1990) procedures (methods 976.05 and 964.06, respectively). Indicator concentrations of samples were measured according to Van Keulen and Young (1977). The data on gross energy were determined by
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PHYTASE EFFICACY ON NUTRIENT DIGESTIBILITY IN LAYERS Table 2. Effect of phytase on the performance of Hy-Line brown layers from 23 to 28 wk of age
Item
Feed intake (g/d)
Laying rate (%)
Egg mass (g/d)
Egg weight (g/egg)
Feed conversion ratio (feed:egg; g/g)
Shell hardness (g/cm2)
Shell thickness (mm)
Positive control Negative control Negative control + A1 Negative control + B1 Negative control + C1 SEM
97.43a 94.11b 99.35a 98.72a 96.75a 0.49
77.91ab 72.63b 80.43a 79.82a 81.61a 1.03
40.59a 37.23b 41.87a 40.33a 41.16a 0.01
52.14a 51.35ab 52.11a 50.42b 50.43b 0.22
2.41 2.45 2.38 2.43 2.35 0.03
4857.28a 4319.90b 4639.40ab 4501.84ab 4769.72ab 77.74
0.35bc 0.34c 0.36a 0.37a 0.37a 0.00
Mean values within columns not sharing the same superscript are different (P < 0.05). A, B, and C are phytases, each at 300 phytase units/kg of feed. A is derived from Aspergillus niger, B and C are derived from Escherichia coli. a–c 1
an oxygen bomb calorimeter (Model 1281, Parr Corp., Moline, IL), and Ca by a polarized Zeeman atomic absorption spectrophotometer (Model Z-8200, Hitachi Corp., Tokyo, Japan). Amino acids were determined by an automatic amino acid analyzer (Model S433D, Sykam Corp., Eresing, Germany) after hydrolyzing the samples in 6 M glass-distilled HCl containing 0.1% phenol for 24 h at 110°C in sealed tubes, with 2 replicates per sample. Methionine was determined in samples that had been oxidized with performic acid prior to acid hydrolysis, according to the procedure of Moore (1963), with the exception that the excess performic acid was removed by lyophilization after dilution with water.
Eggshell Quality Measurements Eggshell hardness was measured by using a texture analyzer (Model P25, TA-XTZi Corp., Surrey, United Kingdom). The egg was laid flat on the pan of the texture analyzer and squeezed, and the shell hardness was recorded. Eggshell thickness was measured with a micrometer (Model 232, Zonechain Corp., Shanghai, China) as the average thickness of the rounded end, pointed end, and middle of the egg, excluding the inner membrane.
Statistical Analysis Experimental data were analyzed by the ANOVA module of SPSS 10.0 software (SPSS, 1999). Significant differences were analyzed by Fisher’s least significant differences test at a P < 0.05 level of significance.
RESULTS Feed intake, laying rate, egg mass, and eggshell thickness were significantly different among treatment diets (P < 0.05; Table 2). The reduction in Ca and P levels in diets without phytase supplementation significantly depressed (P < 0.05) production performance, except for the feed conversion ratio. Supplementation of the negative control diet with phytases significantly improved the performance of birds and the eggshell thickness (P < 0.05). Adding phytases to the negative control diet increased feed intake, egg mass, eggshell hardness, and eggshell thickness by 3.8%, 8.0%, 3.9%, 317.1 g/cm, and 0.03 mm,
respectively, but there were no significant differences in laying rate, egg mass, eggshell hardness, and eggshell thickness among the 3 phytase treatments. The reductions in P and Ca in the negative control diet significantly depressed ileal digestible energy by 0.87 MJ/ kg, the digestibility of N by 6.8%, and the digestibility of P by 20.1% compared with the positive control diet (P < 0.05; Table 3). Adding phytases to the negative control diet improved the digestibility of N, Ca, and P (P < 0.05). Adding phytases to the negative control diet did not significantly improve ileal digestible energy. The digestibility of essential amino acids was 4 to 16% lower and that of nonessential amino acids was 5 to 13% lower for the negative control diet than for the positive control diet (P < 0.05; Table 4). Phytase supplementation in the negative control diet improved the digestibility of amino acids by 2 to 8% (P < 0.05), but the digestibility of most amino acids did not reach the level of the positive control diet. The digestibility of Met was improved by 2.9 to 3.3%. Phytase supplementation numerically improved the digestibility of Lys (3.9%), Arg (2.5%), His (5.2%), Phe (4.2%), Leu (3.8%), Ile (16.8%), Thr (7.1%), and Val (6.5%). The digestibility of nonessential amino acids was also numerically improved with Tyr (6.1%), Ser (6.3%), Asp (4.71%), Glu (3.8%), Gly (6.5%), Ala (5.3%), and Pro (2.7%). There was no difference in the digestibility of most amino acids among the 3 phytase treatments, except for Leu and Ala, for which the phytase C treatment had a higher digestibility than the phytase A treatment.
DISCUSSION Reduction of the NPP level from 0.28 to 0.15% significantly depressed egg production and eggshell quality in the current study, indicating that P was the limiting factor in the performance of birds. The reduction of P and Ca in the negative control group also reduced the digestibility of energy, N, and amino acids compared with the positive control group. This may have been caused by interactions among ingredients, such that the high level of soybean oil in the positive control diet may have contributed to better digestion and utilization of other nutrients as a result of the essential fatty acids, although this has not been fully assessed. Singh and Krikorian (1982) also reported that Ca is required for trypsin activation
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LIU ET AL. Table 3. Effect of phytase on the ileal digestibility of nutrients in Hy-Line brown layers from 23 to 28 wk of age Digestible energy [Mcal/kg (MJ/kg)]
Item Positive control Negative control Negative control + A1 Negative control + B1 Negative control + C1 SEM
2.65 2.44 2.39 2.32 2.56 0.03
(11.07)a (10.20)b (9.97)b (9.69)b (10.69)ab
N (%)
Ca (%)
P (%)
74.62a 67.84c 71.93ab 70.27bc 72.36ab 0.61
45.37ab 33.25b —2 37.79ab 48.33a 2.34
50.68a 30.59c 39.29b 41.61ab 44.12ab 1.79
Mean values within columns not sharing the same superscript are different (P < 0.05). A, B, and C are phytases, each at 300 phytase units/kg of feed. A is derived from Aspergillus niger, B and C are derived from Escherichia coli. 2 A dash (—) indicates a missing value. a–c 1
and can indirectly affect dietary protein utilization by reducing the trypsin-mediated activation of other enzymes. Low-Ca and low-P diets were shown to directly or indirectly depress the metabolism of nutrients in broilers (Rutherfurd et al., 2004) and the performance in layers (Punna and Roland, 1999; Boling et al., 2000a,b; Keshavarz, 2000, 2003a,b). Supplementation of the low-Ca and low-P diet with phytases restored the performance of layers compared with the negative control diet. These results are in agreement with those of Panda et al. (2005), Lim et al. (2003), and Roland et al. (2003). Panda et al. (2005) reported that the addition of phytase to corn and soybean diets containing 0.12% NPP improved the egg production and eggshell quality of layers to the level of those fed diets containing 0.18 to 0.30% NPP. However, Rao et al. (2003) reported that adding phytase to a diet with 3.25% Ca and 0.28% NPP did not improve the performance and retention of Ca and P in the bone and serum of White Leghorn layers, suggesting that 0.28% NPP is adequate for laying hens. The comparison of egg production among phytase treatments and with the positive control group in the current study also revealed that phytase numerically
improved egg production. This might have been due to the total nutrient intake, which was higher in the phytase A and B groups but not in the phytase C group compared with the positive control. In addition to the difference in feed intake, phytase can overcome the adverse effects of phytate on endogenous excretion, as shown in broilers, and can reduce the maintenance requirement of chickens fed a phytase diet (Cowieson et al., 2004), resulting in more nutrients for production. Egg weight was not affected by phytase supplementation in the current study, a result supported by Carlos and Edwards (1998) and Jalal and Scheideler (2001). However, several published reports previously demonstrated that supplementation of phytase generally enhanced the egg production of birds, coupled with egg weight (Um and Paik, 1999; Silversides et al., 2006), which was associated with the dietary NPP level (Wu et al., 2006). We also noted that in the current study, the dietary levels of Ca and P and supplementation of phytase significantly improved eggshell thickness, but not eggshell hardness. The increase in shell thickness was not reflected in shell hardness. Although the exact reason is unknown, this result is in agreement with that reported by Um and Paik (1999),
Table 4. Effect of phytase on the ileal digestibility of amino acids in Hy-Line brown layers from 23 to 28 wk of age Amino acid (%)
Positive control
Negative control
Negative control + A1
Negative control + B1
Negative control + C1
SEM
Met Lys Arg His Phe Leu Ile Thr Val Tyr Ser Asp Glu Gly Ala Pro
88.95a 76.49a 86.88a 61.35a 84.94a 83.82ab 76.95a 68.30a 74.17a 81.15a 77.09a 77.79a 84.84a 70.31a 81.89a 79.07a
84.67b 67.68c 80.73c 45.79c 75.46d 76.57d 66.33c 53.11c 62.59c 71.20c 65.36c 67.44c 79.22c 57.65c 71.10d 71.19c
87.92a 71.04b 83.98b 50.53b 80.05b 81.13bc 72.10b 60.53b 68.42b 77.40b 72.00b 72.23b 82.38b 62.87b 74.87c 76.40b
87.78a 72.42b 82.55bc 50.31b 80.96b 81.47ac 72.94b 58.67b 68.84b 78.12b 70.24b 71.34b 83.14b 64.79b 76.80bc 74.03b
87.59a 71.16b 83.30b 52.03b 78.09c 84.49a 73.41b 61.45b 69.94b 78.80b 72.88b 72.44b 83.49ab 64.75b 77.66b 76.00b
0.34 0.59 0.44 1.05 0.60 0.47 0.60 0.96 0.75 0.65 0.75 0.67 0.39 0.83 0.68 0.57
Mean values within rows not sharing the same superscript are different (P < 0.05). A, B, and C are phytases, each at 300 phytase units/kg of feed. A is derived from Aspergillus niger, B and C are derived from Escherichia coli. a–d 1
PHYTASE EFFICACY ON NUTRIENT DIGESTIBILITY IN LAYERS
who suggested that the association between eggshell hardness and thickness was not inevitable. Shell thickness mainly depends on Ca aggregations as calcium carbonate, whereas shell hardness mainly depends on the texture, composed of Ca carbonate, organic materials, and trace minerals (Chowdhury and Smith, 2002; Mabe et al., 2003; Nakano et al., 2003). Supplementation with phytase A, B, and C did not significantly improve the ileal digestible energy, but with the phytase C treatment the digestible energy was numerically improved by 0.49 MJ/kg. An improvement in energy by applying phytase also was reported in broilers by Ravindran et al. (2006) and Cowieson et al. (2006b). Such improvement in the energy value is a reflection of the increase in digestibility of organic nutrients, including protein, fat, and starch. However, the improvement is often small and statistical significance is difficult to reach because of the large variation among individual birds or replications and other systematic experimental error. The supplementation of phytase significantly improved the digestion of amino acids in the current study. Theoretically, the impact of phytate on the digestion of N in birds mainly results from the phytate-protein complexes existing in feedstuffs or being formed de novo in the gastrointestinal tract under acidic conditions, consequently restricting the contact of phytase to its substrate and the digestion of refractory complexes by pepsin (Selle et al., 2000). The addition of phytase in the poultry diet partially prevents the formation of phytate-protein complexes by the prior hydrolysis of phytate, and thus increases the digestibility of protein. In the current study, adding phytase to a low-Ca and low-P corn-, soybean-, and by-product-based diet improved the ileal digestibility of amino acids by approximately 5%, with the magnitude being variable among amino acids. Ravindran et al. (2006) and Cowieson et al. (2006b) also reported that phytase could enhance the digestibility of amino acids by 1.0 to 15.7% in broilers. However, Jalal et al. (1999) and Snow et al. (2003) studied the effect of phytase on the digestibility of amino acids and found inconsistent results. Jalal et al. (1999) reported that the digestibility coefficients of Met, Cys, Ala, and Glu were significantly improved by phytase in a corn-soybean meal diet, but Snow et al. (2003) found that only the digestibility of Cys was significantly affected in a corn-, soybean-, and meat and bone mealbased diet. Importantly, in the current study phytase supplementation improved the digestibility coefficient of Met by 3%. This result is consistent with findings in broilers (Camden et al., 2001; Rutherfurd et al., 2002, 2004; Ravindran et al., 2006). Cowieson et al. (2006a) observed that phytate depressed the digestibility of sulfur amino acids more severely than the digestibility of other amino acids, suggesting that Met + Cys may be affected to a greater extent by phytate than the other amino acids. There were no significant differences in layer performance and in the digestibility of nutrients by layers among the 3 phytase treatments in the current study. It is well understood that phytases derived from different organisms have different biochemical characteristics,
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such as pH profile and resistance to endogenous protease degradation, which determine their bioefficacy (Kumar et al., 2003). Increasing evidence indicates that the bacterial phytases are more effective in poultry. Augspurger et al. (2003) reported that bacterial phytase derived from E. coli liberated more P in broilers than 2 recombinant fungal phytases. Rodriguez et al. (1999) and Igbasan et al. (2000) and Kumar et al. (2003) reported that E. coli phytase was more resistant to pepsin activity than fungal phytases. Although the digestible energy data showed that phytase C (derived from E. coli) tended to be more effective than the other phytases, the results were inconclusive. More studies with diets of a lower P level are required to identify the differences in bioefficacy among these phytases. In summary, the results of the current study demonstrated that supplementation of phytase at 300 phytase units/kg of feed in diets containing a high level of byproducts with a reduced P level restored the performance of layers to the level of a P-adequate positive control. The ileal digestibility of P, N, Ca, and amino acids was improved by phytase compared with the negative control diet, as reported in broilers. The outcomes of this study will enable layer farmers to use phytase more effectively for maximum profitability.
ACKNOWLEDGMENTS The experiment was conducted at Layer Farm, Zhongte Co. Ltd., Cangzhou, China. The authors are grateful to the staff at the Layer Farm for their support during the experiment.
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