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Comparison of 3 phytases on energy utilization of a nutritionally marginal wheat-soybean meal broiler diet
Comparison of 3 phytases on energy utilization of a nutritionally marginal wheat-soybean meal broiler diet D. Wu,∗ S. B. Wu,∗ M. Choct,†,1 and R. A. Swick∗ ∗
School of Environmental and Rural Sciences, The University of New England, Armidale, NSW 2351, Australia; and † Poultry Cooperative Research Centre, Armidale, NSW 2351, Australia replicates with 16 birds per replicate. The birds were reared until d 21 in floor pens with hardwood shavings. Thirty-two birds (8 birds per treatment) were randomly selected to determine heat production and NE (from 25–28 d) following a 3-d acclimatization in the respiratory chambers. Performance results at d 21 showed that supplementation with either of the 3 phytases improved body weight (P < 0.001) and feed intake (P < 0.05), and increased the relative weights of tibia ash (P < 0.05) and toe ash (P < 0.01). Phytases A and B increased the NE value of the diet (P < 0.05). It may be concluded that the negative effects imposed by calcium and available phosphorus down-specification can be compensated by phytase supplementation in general, and intrinsically thermostable phytases improve the ME and NE value. However, phytase did not reduce heat production, heat increment, or increase NE:ME in birds.
Key words: broiler, microbial phytase, net energy, bone characteristics, growth performance 2015 Poultry Science 94:2670–2676 http://dx.doi.org/10.3382/ps/pev222
INTRODUCTION Phytate (IP6) is the main storage form of phosphorus (P) in plants, representing 60 to 70% of plant P (Karimi et al., 2013). However, the phytate P is poorly digested in monogastric animals, and the phytate molecule has the ability to strongly chelate with cations to form insoluble mineral and other complexes with phytate (Woyengo and Nyachoti, 2013). Hence, exogenous phytase has been introduced to catalyze the phytate content in seed-based feedstuffs to release P for animals to metabolize. In the poultry industry, the conventional way to use exogenous phytase is to assign a phosphate matrix to it and use it at an inclusion rate of 500 FTU/kg broiler feed. However, additional benefits have been shown using phytase at higher levels, resulting in the term “super-dosing” (Cowieson et al., 2011). Super-dosing phytase at levels from 1,000
C 2015 Poultry Science Association Inc. Received December 11, 2014. Accepted June 24, 2015. 1 Corresponding author: [email protected]
FTU/kg to 1,500 FTU/kg is an attempt to achieve extra-phosphoric effects, and this has attracted intensive attention (Cowieson et al., 2011; Walk et al., 2013). The use of phytase at such an unconventionally high dosage cannot only compensate any deficiency of available P by releasing P from hydrolyzed phytate, but also benefit growth and performance by denaturing phytate, rendering it less detrimental as a chelating antinutrient. Phytate is a potent anti-nutrient that impedes digestion even with a relatively low level in the diet (Woyengo and Nyachoti, 2013). It has been postulated that soluble phytate may hinder the effect of duodenal disaccharidases, retard the activity of Na+ K+ -ATPase, stimulate the endogenous secretion of sialic acid, augment ileal endogenous amino acid flow, and increase endogenous sodium losses (Cowieson et al., 2008; Liu et al., 2008). Therefore, the use of unconventionally high dosages of phytase to achieve near-complete IP6 hydrolysis results in performance improvement that is not solely due to phosphate release. The traditional way to evaluate the energy level of a broiler diet is to measure apparent metabolizable energy (AME). According to previous research, the
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ABSTRACT The net energy (NE) value may be a better measure than apparent metabolizable energy (ME) of the effect of supplemental phytase on energy utilization in broilers. The present study was conducted to assess the impact of 3 microbial phytases supplemented at an unconventionally high level (1,000 FTU/kg feed) on performance and NE of broilers using the indirect calorimetric method (IC). Four treatments included: 1) Control, formulated to be deficient in ME (12.35 MJ/kg in the starter diet; 12.56 MJ/kg in the grower diet), calcium (0.72% in the starter diet; 0.60% in the grower diet), and available phosphorus (0.25% in the starter diet; 0.20% in the grower diet); 2) control + intrinsically thermostable phytase A; 3) control + intrinsically thermostable phytase B; and 4) control + coated phytase C. A completely randomized design was employed. A total of 384 male broiler chicks were used, and each treatment had 6
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MATERIALS AND METHODS Animal Husbandry, Diet, and Experimental Design A total of 384 1-day-old male broiler chicks (Ross 308) were purchased from the Baiada Hatchery, Tamworth, New South Wales and were placed in 24 floor pens housed in the University of New England’s animal facility in Armidale, Australia. The study was approved by the Animal Ethics Committee of the University of New England and was designed to comply with the Australian code of practice for the care and use of animals for scientific purposes. The chicks were randomly allocated to 4 dietary treatments: 1) Control, formulated to be deficient in ME (12.35 MJ/kg in the starter diet; 12.56 MJ/kg in the grower diet), available P (0.25% in the starter diet; 0.20% in the grower diet) and Ca (0.72% in the starter diet; 0.60% in the grower diet); 2) control supplemented with phytase A (Quantum Blue, manufactured by AB Vista Feed Ingredients, Marlborough, UK); 3) control supplemented with phytase B (Quantum XT, produced by AB Vista Feed Ingredients, Marlborough, UK); and 4) control supplemented with phytase C (Phyzyme TPT, manufactured by Danisco Animal Nutrition, Marlborough, UK). All the 3 phytase preparations were Escherichia coli–derived 6-phytase, and all applied at a rate of 1,000 FTU/kg of feed. Phytases A and C were supplemented as granular formulations, while phytase B was applied in liquid form. Each treatment included 6 replicates of 16 birds per replicate. Feed and water were offered to birds for
Table 1. Ingredient composition and calculated nutrient levels of basal diet. Ingredients4 Wheat Soybean meal Canola meal (solvent) Meat meal Canola oil Limestone Salt Sodium bicarbonate L-lysine HCl DL-methionine L-threonine Choline Cl 70% Mineral premix1 Vitamin premix2 Salinomycin (12%) Zn bacitracin (15%) Xylanase (Porzyme 93010, Dupont) Calculated nutrient level4 ME poultry (MJ/kg) Crude protein Calcium Phosphorus, NPP5 Phosphorus, total Sodium Choline (mg/kg) Dig3 lysine Dig methionine Dig methionine + cysteine Dig tryptophan Dig isoleucine Dig arginine Dig threonine Dig valine
Starter 60.3 24.2 9.6 1.7 1.8 1.1 0.31 0.20 0.27 0.21 0.03 0.11 0.075 0.05 0.05 0.033 0.005
Grower 59.1 25.0 10.0 1.0 3.1 0.9 0.21 0.20 0.10 0.16 0.00 0.04 0.075 0.05 0.05 0.033 0.005
12.35 12.56 23.1 22.5 0.75 0.60 0.25 0.20 0.54 0.52 0.22 0.18 1850 1470 1.20 1.10 0.44 0.48 0.84 0.80 0.19 0.27 0.78 0.85 1.26 1.29 0.74 0.70 0.92 0.95
1 Mineral mix supplied the following per kilogram of diet: Mn, 120 mg; Zn, 100 mg; Fe, 40 mg; Cu, 16 mg; Se, 0.3 mg; I, 1.25 mg. 2 Vitamins supplied the following amounts per kilogram of diet: vitamin A, 12,000 IU; cholecalciferol, 5,000 IU; vitamin E, 75 IU; vitamin K, 3 mg; vitamin B12 , 16 mcg; riboflavin, 8 mg; pantothenic acid, 13 mg; nicotinic acid, 55 mg; folic acid, 2 mg; biotin, 0.2 mg. 3 Dig = digestible. 4 Percent unless otherwise indicated. 5 Non-phytate phosphorus.
ad libitum consumption. The lighting program was 24 h of light during the first 3 d, 23 h of light from 4 d to 7 d, 20 h of light from 8 d to 10 d, and 18 h of light thereafter until the end of the study. Birds were fed in 2 phases: starter (0–21 d) and finisher (21–28 d). The basal diet was formulated to be deficient in ME, available P, and Ca. The ingredient composition of the basal diet and nutrient levels are shown in Table 1. A basal diet with the nutrient composition of the control was mixed first and then evenly divided into 4 groups, with the first group being the control, and the remaining 3 were treated with different phytases according to the experimental design. The starter diets were supplied in mash form, whereas the grower diets were pelleted. Pelleting temperature was maintained between 65 and 70◦ C. Pellets were spread onto a cool clean surface to a depth of 3 cm for cooling. After cooling, the liquid form, phytase B was applied as a diluted spray
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influence of phytase on dietary AME is inconsistent (Ravindran et al., 1999a; Shirley and Edwards, 2003; Pirgozliev et al., 2011). Since the AME system does not take into account the energy partitioned to production and heat increment, it has been suggested that the net energy (NE) system may offer an advantage in estimating energy utilization in poultry diets. Pirgozliev and Bedford (2013) found that dietary net energy for production (NEp) had a higher correlation to performance when compared to AME. This suggests that NE may be a more sensitive way to evaluate broiler response to phytase supplementation. Similar findings were also reported by Olukosi et al. (2008). Previous research focusing solely on the effect of phytase on AME may have underestimated the full value of exogenous phytase in the improvement of broiler performance. It was hypothesized that supplementation of phytase at an unconventionally high level (1,000 FTU/kg feed) improves broiler performance by limiting heat increment and producing a higher NE:ME ratio. Thus, the objective of the current study was to investigate the effects of 3 supplemental phytases, all used at a high dosage (1,000 FTU/kg), on growth performance, NE, relative digestive tract development, and bone mineralization in broiler chickens.
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WU ET AL. Table 2. The analyzed nutrients and phytase activity of diets. Treatment Starter
Grower
Control Control Control Control Control Control Control Control
(basal) + phytase + phytase + phytase (basal) + phytase + phytase + phytase
Calcium,%
P, total,%
Analyzed phytase activity in feed samples (FTU/kg)
0.84
0.55
0.63
0.54
74 823 794 1,072 467 1,552 1,480 1,649
A B C A B C
Sample Collection On d 21, 2 birds per pen were removed, weighed, and euthanized by cervical dislocation. The digestive tract, from the proventriculus to small intestine, was carefully excised. The weight of proventriculus plus gizzard was recorded. The weight of the small intestine (from the beginning of duodenum to the ileocecal junction) was obtained after the contents inside were emptied by gentle digital expression. The whole left leg of those 2 birds per pen was excised from the fresh carcass, sealed in plastic bags to minimize moisture loss, and then stored at –20◦ C until they were used for the determination of bone characteristics.
Net Energy Measurement At 22 d of age, 32 birds (8 birds from each treatment) were randomly selected and transferred from the rearing floor pens to 16 calorimeter chambers in a temperature-controlled room for a 3-d acclimatization. The birds were given their respective test diets, each with 4 replicates of 2 birds housed in a closed-circuit calorimeter chamber. The closed-circuit calorimeter chambers were newly constructed by the workshop of University of New England, and the design of the closed-circuit calorimetric chambers and the measurement of O2 consumptions and CO2 release by the birds using the chamber have been described previously (Swick et al., 2013). Briefly, the chamber was equipped with a wire-mesh cage, chamber air circulated through KOH to absorb CO2 and silica gel to remove moisture. An oxygen cylinder was fitted to replenish
O2 through pressure-controlled solenoid valve, and oxygen consumption was measured gravimetrically. The recovery of CO2 was analyzed by barium carbonate precipitation (Annison and White, 1961). Air-conditioning units were used maintain constant temperatures of 22 ± 1◦ C inside the room and 24 ± 1◦ C inside the chambers. Light was controlled to achieve 15 lux at the bird level. The heat production (HP) was estimated using the Brouwer equation (Brouwer, 1965): kcal total heat = 3.866 × L of oxygen consumed + 1.200 × L of CO2 expired. The AME was measured using the total collection method (Bourdillon et al., 1990). Excreta were collected and feed consumption recorded each day. The excreta from each chamber were thoroughly mixed into slurry using a blender and sub-samples taken for gross energy (GE) analysis. The experimental diets and excreta were analyzed R for GE using an adiabatic bomb calorimeter (IKA WERKE, C7000, GMBH and Co., Staufen, Germany). Benzoic acid was used as a standard. The respiratory quotient (RQ) of the 3-d run was calculated as the ratio of the volume of CO2 expired to the volume of O2 consumed. Heat increment (HI) was calculated by deducting fasting HP from total HP. The value of 450 kJ/kg BW0.70 per bird per day was applied as fasting HP, which corresponds to the asymptotic HP (at zero activity) during a 24-h fast, as proposed by (Noblet et al. 2010a, 2010b). NE was calculated as ME intake minus HI divided by consumption of feed on an as-is basis.
Tibia Ash, Toe Ash, and Tibia Bone Breaking Strength The legs of the 2 sacrificed birds were thawed, and tibia bones were severed and defleshed without boiling; the middle toe was clipped and cleaned of any waste material such as litter and excrement. Skin, flesh, and toenail of the middle toe were kept intact. The tibias and toes were dried at 105◦ C for 24 h and placed in a desiccator, and the weights were recorded. Tibia breaking strength was measured using an Instron instrument (Model 1011 Instron Universal Testing Machine, Instron Corp., Canton, MA). The instrument was
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(250× dilutions) of the received product. Each batch of pellets was sprayed evenly twice. The analyzed phytase activity and the inclusion level of Ca and total P of feed samples is shown in Table 2. One phytase unit is defined as the amount of enzyme required to release 1 μmol of inorganic P/min from sodium phytate at 37 ± 0.1◦ C and pH 5.5. Dietary phytase levels were analyzed by Pacific Lab Services (Singapore) according to method 2000.12 ( AOAC International, 2005). Dietary inclusion level of Ca and total P were analyzed using inductively coupled plasma optical emission spectrometer, and perchloric acid and hydrogen peroxide were used for digestion (Anderson and Henderson, 1986).
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MICROBIAL PHYTASE, NET ENERGY AND BONE CHARACTERISTICS Table 3. Responses of growth performance and relative organ weights from 0–21 d to 3 phytases1 . Treatment
BW g
Feed intake g
Feed/ gain g/g
Livability %
Gizzard + Proventriculus2
Small Intestine2
Control Control + phytase A Control + phytase B Control + phytase C SEM P>F
761b 853a 868a 859a 12.4 < 0.001
1,077b 1,223a 1,190a 1,164a 17.1 < 0.01
1.512 1.518 1.447 1.433 0.02 0.53
96.88 100 100 98.96 0.81 0.51
14.60 13.77 13.98 14.13 0.34 0.86
28.25a 24.11b 24.27b 26.68a,b 0.57 < 0.05
means in a column not sharing a common superscript differ (P < 0.05). The values are the means of 6 replicates from each treatment. 2 Relative weight (g/kg body weight). a,b 1
Statistical Analysis The data were analyzed using the SPSS 21.0 software program (SPSS Inc., Chicago, IL). One-way ANOVA was used to determine the difference among the treatments. If significant treatment effects were observed, the differences of means between treatments were determined using the least significant difference test. Differences were considered significant at P < 0.05.
RESULTS Table 1 shows expected and recovered phytase from experimental mixed feeds. Table 3 shows the results of the 21-d performance experiment. The supplementation of the 3 microbial phytases increased the average BW at 21 d (P < 0.001). The addition of phytases A, B, and C improved the average BW by 12.1, 14.1, and 12.9%, respectively, compared with the birds fed the control diet. A similar trend was observed for feed intake. Feed intake of birds fed the diets supplemented with all 3 phytases was significantly increased by 13.6, 10.5, and 8.1%, respectively (P < 0.01). None of the phytases had any effect on FCR (P >0.05). Phytase had no impact on livability (P > 0.05). Supplementation of phytases A and B reduced the relative weight of the small intestine compared to the control by 14.7 and 14.1%, respectively (P < 0.05). Results shown in Table 4 indicate that during the 3-d period (d 25–27) of measurements of respiratory gases in chambers, no difference in RQ, performance, HP, or NE:ME ratio was observed between treatments (P > 0.05). NE per kg of diet was significantly increased by phytases A and B (P < 0.05) by 5.6 and 5.1%, respectively. It was also observed that phytase B tended to improve MEI (P = 0.07), and both phytases A and B tended to improve ME in comparison with phytase C (P = 0.09).
Phytase supplementation increased the relative weights of tibia ash and toe ash (Table 5), with phytase B improving the relative weight of tibia ash by 11.0% (P < 0.01) and phytase A numerically (P = 0.097) increasing the relative weight of tibia ash by 6.2%. Similarly, both phytases A and B resulted in, respectively, 18.3 and 17.6% higher relative weight of toe ash compared to the control (P < 0.001). However, phytase supplementation had no effect (P > 0.05) on tibia bone breaking strength.
DISCUSSION The study used diets formulated to be deficient in available P, Ca, and ME to examine if 3 different E. coliderived phytases elicited energy responses differently. Phytase recovery from diets is shown in Table 1. The results suggest reasonable expected recovery. Differences in assayed phytase levels in the non-supplemented starter and grower control basal diets were observed, and supplemented starter diets were more variable than grower diets. The starter diets had higher limestone, salt, and meat meal inclusion than the grower diets. It is possible that these minerals may have reduced the activity of endogenous phytase from wheat in the starterdiet assay. Feeding study results indicated that supplementation with intrinsically thermostable phytases A and B did not support the hypothesis of improving performance through reduced HI of diets. Although NE was increased in diets supplemented with phytase A and B, no effect of these 2 phytases was observed on HI, which demonstrated that improved performance mainly originated from increased ME concentration in the diets induced by phytases A and B. When energy responses to phytase supplementation are measured using the AME system, the results are often equivocal (Tejedor et al., 2001; Shirley and Edwards, 2003; Cowieson and Adeola, 2005; Chung et al., 2013). But the net-energy system has been considered a more refined way of estimating energy partitioning and utilization in animals, as this system takes into account total HP and HI due to digestion and metabolism of feed (Swick et al., 2013). The current study found that the dietary inclusion of the 2 intrinsically thermostable phytases increased dietary NE, which was in agreement
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equipped with 50-kg-load cell with a crosshead speed of 50 mm/min. Tibias were supported on a 3.35-cm span (Shafer et al., 2001). Moisture-free tibia ash and toe ash were determined by ashing in ceramic crucibles for 24 h at 615◦ C. Tibia ash and toe ash were expressed as the percentages of tibia ash and toe ash relative to tibia and toe dry weights.
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WU ET AL. Table 4. Energy partition and performance responses to 3 different phytases in broilers during 25 and 28 d of age1 . Control no phytase
Phytase A
Phytase B
Phytase C
SEM
P>F
1,402 88.0 1.595 1,368 851 401 1.02 13.87 9.80b 70.7
1,404 92.8 1.560 1,460 851 401 1.04 14.26 10.35a 72.6
1,391 97.9 1.510 1,503 866 416 1.02 14.23 10.30a 72.3
1,386 86.4 1.618 1,354 825 375 1.02 13.65 9.86b 72.3
13.9 2.4 0.023 23.7 7.2 7.2 0.004 0.103 0.088 0.397
0.972 0.407 0.456 0.068 0.267 0.267 0.354 0.086 0.024 0.288
BW, g ADG, g Feed conversion ratio, g/g MEI, kJ/kg BW0.70 HP, kJ/kg BW0.70 HI, kJ/kg BW0.7 RQ ME, kJ/g feed DM NE, kJ/g feed DM NE:ME (%)
means in a row not sharing a common superscript differ (P < 0.05). The values are the means of 4 replicates from each treatment.
a,b 1
Table 5. Bone mineralization changes in response to 3 phytases in broilers during 0–21 d of age1 .
Control Control + phytase A Control + phytase B Control + phytase C SEM P>F
Tibia ash (%) b
35.6 37.8a,b 39.5a 37.2a,b 0.5 0.043
Toe ash (%) b
11.1 13.1a 13.1a 12.0b 0.2 0.001
Bone breaking strength (N) 82.2 72.8 90.5 66.2 3.5 0.055
means in a column not sharing a common superscript differ (P < 0.05). The values are the means of 6 replicates from each treatment.
a,b 1
with previous reports (Olukosi et al., 2008; Pirgozliev and Bedford, 2013). However, the improvement of dietary NE largely originated from the increased ME of the diets supplemented with phytase A and phytase B. This contradicts the hypothesis of lower HP and HI as a result of phytase supplementation. Furthermore, there was no effect of phytase on the ratio of NE to ME. In the current study, a tendency for phytase supplementation to improve ME was observed in the following order: A > B > control = C (P < 0.09). This energy effect of phytase is likely due to improved availability of energy-yielding components in the diet and perhaps improved amino acid digestibility. Phytase has been shown to denature Ca-phytate complexes, which are involved in the formation in the gut lumen of metallic soaps that limit energy utilization from fat (Ravindran et al., 2001). There is much evidence that exogenous phytase is capable of increasing amino acid digestibility from a variety of diet types (Namkung and Leeson, 1999; Camden et al., 2001; Ravindran et al., 2001). Phytate is an integral component of the cell-wall matrix in wheat (Frolich, 1990), and supplementation with phytase may increase the accessibility of digestive enzymes to their respective substrates, thus increasing the ME value of diets. Phytase may also reduce endogenous losses, thereby increasing ME. There is large variation in endogenous losses among birds fed different feed ingredients (Siriwan et al., 1993; Ravindran et al., 2006). Feed intake, enzyme supplementation, and other feed additives can have an effect on endogenous losses (Angkanaporn
et al., 1994; Cowieson et al., 2006; Pirgozliev et al., 2008). Ravindran et al. (2006) found phytate increased Na losses by 60% in ad libitum-fed broiler chickens, while phytase reduced these losses by 66%. Similar effects of phytase were reported on sialic acid secretion by Cowieson et al. (2004) and Pirgozliev et al. (2007). The reduction in endogenous losses would be expected to lead to a reduction of the energy required for maintenance, thus allowing a greater proportion of metabolizable energy to be partitioned towards growth. Nevertheless, it was unexpected that the coated phytase failed to deliver the same result on dietary ME and NE as the 2 intrinsically thermostable counterparts. The discrepancy on effectiveness may be related to the rate of reaction of phytate degradation. The mean retention time for digesta within the gastrointestinal tract is less than 3.5 h (Hughes, 2008), and only a small portion of that time is spent in the crop, gizzard, and proventriculus, where phytate is soluble and available for dephosphorylation and reaction with phytase. In this regard, it is possible that the intrinsically thermostable phytases, without coating, are more soluble in the upper gastrointestinal tract and are more bioactive in this region than coated phytase. The use and selection of phytase product type would, of course, depend on whether it is intended for pre- or post- pellet application. Dietary phytate has been considered as an antinutrient as it forms complexes with minerals, starch, and protein, rendering them less available for monogastric animals (Sebastian et al., 1996; Ravindran et al., 1999b; Shelton and Southern, 2006). Monogastric
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Treatment
MICROBIAL PHYTASE, NET ENERGY AND BONE CHARACTERISTICS
especially protein and lipids, so there is a chance that the fixed fasting HP did not represent the true fasting HP in birds fed diets with phytase. Therefore, it is recommended to measure fasting HP directly in future trials when exogenous enzymes are treated. It is concluded that supplemental microbial phytase can beneficially influence growth performance and bone mineralization in ME-, Ca-, and available P-deficient, wheat-based diets. This study has shown that while dietary inclusion of intrinsically thermostable phytases at dose levels of 1,000 FTU/kg feed have the capacity to improve dietary ME and NE. This was a result of increased MEI and was not caused by reduced HI. None of the examined phytases lowered HP or HI, and none increased the ratio of NE to ME.
REFERENCES Anderson, D. L., and L. J. Henderson. 1986. Sealed chamber digestion for plant nutrient analysis. Agron. J. 78:937–938. Angkanaporn, K., M. Choct, W. L. Bryden, E. F. Annison, and G. Annison. 1994. Effects of wheat pentosans on endogenous aminoacid losses in chickens. J. Sci. Food Agric. 66:399–404. Annison, E. F., and R. R. White. 1961. Glucose utilization in sheep. Biochem. J. 80:162–169. AOAC International. 2005. Official methods of analysis. 18th ed., AOAC International, Arlington, VA. Applegate, T. J., R. Angel, and H. L. Classen. 2003. Effect of dietary calcium, 25-hydroxycholecalciferol, or bird strain on small intestinal phytase activity in broiler chickens. Poult. Sci. 82:1140–1148. Bourdillon, A., B. Carre, L. Conan, M. Francesch, M. Fuentes, G. Huyghebaert, W. Janssen, B. Leclercq, M. Lessire, J. McNab, M. Rigoni, and J. Wiseman. 1990. European reference method of invivo determination of metabolizable energy in poultry: reproducibility, effect of age, comparison with predicted values. Br. Poult. Sci. 31:567–576. Brouwer, E. 1965. Report of subcommittee on constants and factors. Proc. the 3rd symposium on energy metabolism, European Federation of Animal Science Publication 11. (Ed. KL Blaxter). 441–443. Camden, B. J., P. C. H. Morel, D. V. Thomas, V. Ravindran, and M.R. Bedford. 2001. Effectiveness of exogenous microbial phytase in improving the bioavailabilities of phosphorus and other nutrients in maize-soya-bean meal diets for broilers. Anim. Sci. 73:289–297. Chung, T. K., S. M. Rutherfurd, D. V. Thomas, and P. J. Moughan. 2013. Effect of two microbial phytases on mineral availability and retention and bone mineral density in low-phosphorus diets for broilers. Br. Poult. Sci. 54:362–373. 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., and O. Adeola. 2005. Carbohydrases, protease, and phytase have an additive beneficial effect in nutritionally marginal diets for broiler chicks. Poult. Sci. 84:1860–1867. Cowieson, A. J., M. Hruby, and E. E. M. Pierson. 2006. Evolving enzyme technology: impact on commercial poultry nutrition. Nutr. Res. Rev. 19:90–103. Cowieson, A. J., P. H. Selle, and V. Ravindran. 2008. Influence of dietary phytic acid and source of microbial phytase on ileal endogenous amino acid flows in broiler chickens. Poult. Sci. 87:64– 64. Cowieson, A. J., P. Wilcock, and M. R. Bedford. 2011. Super-dosing effects of phytase in poultry and other monogastrics. Worlds Poult. Sci. J. 67:225–235. Esteve-Garcia, E., J. Brufau, A. Perez-Vendrell, A. Miquel, and K. Duven. 1997. Bioefficacy of enzyme preparations containing betaglucanase and xylanase activities in broiler diets based on barley or wheat, in combination with flavomycin. Poult. Sci. 76:1728– 1737.
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animals do not produce enough endogenous phytase (Maenz and Classen, 1998; Applegate et al., 2003) and, as a result, exogenous phytase is required in the diets to alleviate the detrimental effects of phytate. In the current study, the phytase-supplemented groups all had substantially higher average BW and feed intake compared with the control, but had little impact on FCR. P deficiency in the diet generally compromises broilers’ appetite, lowers the blood P level, decreases the circulating levels of growth hormone (Parmer et al., 1987) and consequently reduces the rate of weight gain. It has been reported that phytase-improved feed intake in the birds fed available P-deficient diets, and thus weight gain was increased but feed efficiency did not show improvement (Shaw et al., 2010; Karimi et al., 2013). The results of this experiment agree with this mode of action of phytase. The data in the present study showed little impact of phytase addition on HP or HI. However, birds had reduced small intestine size in the treatments supplemented with phytases A and B. According to Spratt et al. (1990), although the liver and the gastrointestinal tract account for approximately 3% of the bodyweight of a hen, they may consume up to 26% of the fasting HP, and the total cost of maintenance may take up to 75% of total fasting HP. Previous studies (EsteveGarcia et al., 1997; Wu et al., 2004) showed that the use of exogenous enzymes often reduced the size of the energetically demanding organs, such as the pancreas and the gut. In the present study, the reduced small intestine size observed in the groups supplemented with phytases A and B is in accord with the earlier findings. However, phytase addition in this study did not have any impact on heat increment or HP. This was unexpected, as it was hypothesized that less energy would be consumed by a smaller gastrointestinal tract and result in lower HP. The likely explanation of the lack of effect on HP in the phytase-supplemented groups is that the enzyme increased the energy need for maintenance of skeletal muscles as well as the energy expenditure associated with the absorption of some energydependent active transported nutrients and minerals (Kies et al., 2005). Considering some phytase addition has increased the relative weight of tibia ash (phytase B, by 11.0%) and toe ash (phytase A, by 18.3%; phytase B, by 17.6%) in the present study, it may be postulated that a higher amount of heat was generated while more P or Ca was released, absorbed, and utilized in the phytase-supplemented treatments. Moreover, a fixed figure for fasting HP (450 kJ/kg BW0.70 per bird per day) was applied in the current study, based on the findings of (Noblet et al. 2010a, 2010b), which suggests that fasting HP in 0.5 to 3.0 kg broilers is proportional to BW0.70 with values ranging between 420 and 450 kJ/kg BW0.70 /d. However, from a practical point of view, the fasting HP figure may be fully justified in broilers fed diets with similar chemical characteristics, but phytase supplementation may alter fasting HP by changing the way broilers digest and utilize nutrients,
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WU ET AL. Ravindran, V., P. C. H. Morel, G. G. Partridge, M. Hruby, and J. S. Sands. 2006. Influence of an Escherichia coli-derived phytase on nutrient utilization in broiler starters fed diets containing varying concentrations of phytic acid. Poult. Sci. 85:82–89. Ravindran, V., P. H. Selle, and W. L. Bryden. 1999b. Effects of phytase supplementation, individually and in combination, with glycanase, on the nutritive value of wheat and barley. Poult Sci. 78:1588–1595. Ravindran, V., P. H. Selle, G. Ravindran, P. C. H. Morel, A. K. Kies, and W. L. Bryden, 2001. Microbial phytase improves performance, apparent metabolizable energy, and ileal amino acid digestibility of broilers fed a lysine-deficient diet. Poult. Sci. 80:338– 344. Sebastian, S., S. P. Touchburn, E. R. Chavez, and P. C. Lague. 1996. The effects of supplemental microbial phytase on the performance and utilization of dietary calcium, phosphorus, copper, and zinc in broiler chickens fed corn-soybean diets. Poult. Sci. 75:729– 736. Shafer, D. J., R. P. Burgess, K. A. Conrad, J. F. Prochaska, and J. B. Carey. 2001. Characterization of alkaline hydroxide-preserved whole poultry as a dry byproduct meal. Poult. Sci. 80:1543–1548. Shaw, A. L., J. P. Blake, and R. W. Gordon. 2010. Evaluation of commercial phytase enzymes on performance and tibia-breaking strength of male broiler chicks. J. Appl. Poult. Res. 19:415– 421. Shelton, J. L., and L. L. Southern. 2006. Effects of phytase addition with or without a trace mineral premix on growth performance, bone response variables, and tissue mineral concentrations in commercial broilers. J. Appl. Poult. Res. 15:94–102. Shirley, R. B., and H. M. Edwards. 2003. Graded levels of phytase past industry standards improves broiler performance. Poult. Sci. 82:671–680. Siriwan, P., W. L. Bryden, Y. Mollah, and E. F. Annison. 1993. Measurement of endogenous amino-acid losses in poultry. Br. Poult. Sci. 34:939–949. Spratt, R. S., B. W. McBride, H. S. Bayley, and S. Leeson. 1990. Energy-metabolism of broiler breeder hens .2. contribution of tissues to total heat-production in fed and fasted hens. Poult. Sci. 69:1348–1356. Swick, R. A., S. B. Wu, J. J. Zuo, N. Rodgers, M. R. Barekatain, and M. Choct. 2013. Implications and development of a net energy system for broilers. Anim. Prod. Sci. 53:1231–1237. Tejedor, A. A., L. F. T. Albino, H. S. Rostagno, and F. M. Vieites. 2001. Effect of phytase suplementation on the performance and heal digestibility of nutrients. Rev. Bras. Zootecn. 30:802– 808. Walk, C. L., M. R. Bedford, T. S. Santos, D. Paiva, J. R. Bradley, H. Wladecki, C. Honaker, and A. P. McElroy. 2013. Extraphosphoric effects of superdoses of a novel microbial phytase. Poult. Sci. 92:719–725. Wu, Y. B., V. Ravindran, D. G. Thomas, M. J. Birtles, and W. H. Hendriks. 2004. Influence of phytase and xylanase, individually or in combination, on performance, apparent metabolisable energy, digestive tract measurements and gut morphology in broilers fed wheat-based diets containing adequate level of phosphorus. Br. Poult. Sci. 45:76–84. Woyengo, T. A., and C. M. Nyachoti. 2013. Review: anti-nutritional effects of phytic acid in diets for pigs and poultry — current knowledge and directions for future research. Can. J. Anim. Sci. 93:9–21.
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Frolich, W., 1990. Chelating properties of dietary fiber and phytate. The role for mineral availability. Pages 83–93 in: New Developments in Dietary Fiber. I. Furda, and C. J. Brine, ed. Plenum Press, New York, NY. Hughes, R. J. 2008. Relationship between digesta transit time and apparent metabolisable energy value of wheat in chickens. Br. Poult. Sci. 49:716–720. Karimi, A., C. Coto, F. Mussini, S. Goodgame, C. Lu, J. Yuan, M. R. Bedford, and P. W. Waldroup. 2013. Interactions between phytase and xylanase enzymes in male broiler chicks fed phosphorusdeficient diets from 1 to 18 days of age. Poult. Sci. 92:1818–1823. Kies, A. K., W. J. J. Gerrits, J. W. Schrama, M. J. W. Heetkamp, K. L. van der Linden, T. Zandstra, and M. W. A. Verstegen. 2005. Mineral absorption and excretion as affected by microbial phytase, and their effect on energy metabolism in young piglets. J. Nutr. 135:1131–1138. Liu, N., Y. J. Ru, F. D. Li, and A. J. Cowieson. 2008. Effect of diet containing phytate and phytase on the activity and messenger ribonucleic acid expression of carbohydrase and transporter in chickens. J. Anim. Sci. 86:3432–3439. Maenz, D. D., and H. L. Classen. 1998. Phytase activity in the small intestinal brush border membrane of the chicken. Poult. Sci. 77:557–563. Namkung, H., and S. Leeson. 1999. Effect of phytase enzyme on dietary nitrogen-corrected apparent metabolizable energy and the ileal digestibility of nitrogen and amino acids in broiler chicks. Poult. Sci. 78:1317–1319. Noblet, J., S. Dubois, E. Labussiere, B. Carre, and J. van Milgen. 2010a. Metabolic utilization of energy in monogastric animals and its implementation in net energy systems. Proc. EAAP Int. Symp. Energy Protein Metab. Nutr. 3:573–582. Noblet, J., J. van Milgen, and S. Dubois. 2010b. Utilisation of metabolisable energy of feeds in pigs and poultry: interest of net energy systems? Proc. Aust. Poult. Sci. Symp. 21:26–35. Olukosi, O. A., A. J. Cowleson, and O. Adeola. 2008. Energy utilization and growth performance of broilers receiving diets supplemented with enzymes containing carbohydrase or phytase activity individually or in combination. Br. J. Nutr. 99:682–690. Parmer, T. G., L. B. Carew, F. A. Alster, and C. G. Scanes. 1987. Thyroid-function, growth-hormone, and organ growth in broilers deficient in phosphorus. Poult. Sci. 66:1995–2004. Pirgozliev, V., and M. R. Bedford. 2013. Energy utilisation and growth performance of chicken fed diets containing graded levels of supplementary bacterial phytase. Br. J. Nutr. 109: 248–253. Pirgozliev, V., M. R. Bedford, T. Acamovic, P. Mares, and M. Allymehr. 2011. The effects of supplementary bacterial phytase on dietary energy and total tract amino acid digestibility when fed to young chickens. Br. Poult. Sci. 52:245–254. Pirgozliev, V., T. C. Murphy, B. Owens, J. George, and M. E. E. McCann. 2008. Fumaric and sorbic acid as additives in broiler feed. Res. Vet. Sci. 84:387–394. Pirgozliev, V., O. Oduguwa, T. Acamovic, and M. R. Bedford. 2007. Diets containing Escherichia coli-derived phytase on young chickens and turkeys: Effects on performance, metabolizable energy, endogenous secretions, and intestinal morphology. Poult. Sci. 86:705–713. Ravindran, V., S. Cabahug, G. Ravindran, and W. L. Bryden. 1999a. Influence of microbial phytase on apparent ileal amino acid digestibility of feedstuffs for broilers. Poult. Sci. 78:699–706.
School of Environmental and Rural Sciences, The University of New England, Armidale, NSW 2351, Australia; and † Poultry Cooperative Research Centre, Armidale, NSW 2351, Australia replicates with 16 birds per replicate. The birds were reared until d 21 in floor pens with hardwood shavings. Thirty-two birds (8 birds per treatment) were randomly selected to determine heat production and NE (from 25–28 d) following a 3-d acclimatization in the respiratory chambers. Performance results at d 21 showed that supplementation with either of the 3 phytases improved body weight (P < 0.001) and feed intake (P < 0.05), and increased the relative weights of tibia ash (P < 0.05) and toe ash (P < 0.01). Phytases A and B increased the NE value of the diet (P < 0.05). It may be concluded that the negative effects imposed by calcium and available phosphorus down-specification can be compensated by phytase supplementation in general, and intrinsically thermostable phytases improve the ME and NE value. However, phytase did not reduce heat production, heat increment, or increase NE:ME in birds.
Key words: broiler, microbial phytase, net energy, bone characteristics, growth performance 2015 Poultry Science 94:2670–2676 http://dx.doi.org/10.3382/ps/pev222
INTRODUCTION Phytate (IP6) is the main storage form of phosphorus (P) in plants, representing 60 to 70% of plant P (Karimi et al., 2013). However, the phytate P is poorly digested in monogastric animals, and the phytate molecule has the ability to strongly chelate with cations to form insoluble mineral and other complexes with phytate (Woyengo and Nyachoti, 2013). Hence, exogenous phytase has been introduced to catalyze the phytate content in seed-based feedstuffs to release P for animals to metabolize. In the poultry industry, the conventional way to use exogenous phytase is to assign a phosphate matrix to it and use it at an inclusion rate of 500 FTU/kg broiler feed. However, additional benefits have been shown using phytase at higher levels, resulting in the term “super-dosing” (Cowieson et al., 2011). Super-dosing phytase at levels from 1,000
C 2015 Poultry Science Association Inc. Received December 11, 2014. Accepted June 24, 2015. 1 Corresponding author: [email protected]
FTU/kg to 1,500 FTU/kg is an attempt to achieve extra-phosphoric effects, and this has attracted intensive attention (Cowieson et al., 2011; Walk et al., 2013). The use of phytase at such an unconventionally high dosage cannot only compensate any deficiency of available P by releasing P from hydrolyzed phytate, but also benefit growth and performance by denaturing phytate, rendering it less detrimental as a chelating antinutrient. Phytate is a potent anti-nutrient that impedes digestion even with a relatively low level in the diet (Woyengo and Nyachoti, 2013). It has been postulated that soluble phytate may hinder the effect of duodenal disaccharidases, retard the activity of Na+ K+ -ATPase, stimulate the endogenous secretion of sialic acid, augment ileal endogenous amino acid flow, and increase endogenous sodium losses (Cowieson et al., 2008; Liu et al., 2008). Therefore, the use of unconventionally high dosages of phytase to achieve near-complete IP6 hydrolysis results in performance improvement that is not solely due to phosphate release. The traditional way to evaluate the energy level of a broiler diet is to measure apparent metabolizable energy (AME). According to previous research, the
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ABSTRACT The net energy (NE) value may be a better measure than apparent metabolizable energy (ME) of the effect of supplemental phytase on energy utilization in broilers. The present study was conducted to assess the impact of 3 microbial phytases supplemented at an unconventionally high level (1,000 FTU/kg feed) on performance and NE of broilers using the indirect calorimetric method (IC). Four treatments included: 1) Control, formulated to be deficient in ME (12.35 MJ/kg in the starter diet; 12.56 MJ/kg in the grower diet), calcium (0.72% in the starter diet; 0.60% in the grower diet), and available phosphorus (0.25% in the starter diet; 0.20% in the grower diet); 2) control + intrinsically thermostable phytase A; 3) control + intrinsically thermostable phytase B; and 4) control + coated phytase C. A completely randomized design was employed. A total of 384 male broiler chicks were used, and each treatment had 6
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MICROBIAL PHYTASE, NET ENERGY AND BONE CHARACTERISTICS
MATERIALS AND METHODS Animal Husbandry, Diet, and Experimental Design A total of 384 1-day-old male broiler chicks (Ross 308) were purchased from the Baiada Hatchery, Tamworth, New South Wales and were placed in 24 floor pens housed in the University of New England’s animal facility in Armidale, Australia. The study was approved by the Animal Ethics Committee of the University of New England and was designed to comply with the Australian code of practice for the care and use of animals for scientific purposes. The chicks were randomly allocated to 4 dietary treatments: 1) Control, formulated to be deficient in ME (12.35 MJ/kg in the starter diet; 12.56 MJ/kg in the grower diet), available P (0.25% in the starter diet; 0.20% in the grower diet) and Ca (0.72% in the starter diet; 0.60% in the grower diet); 2) control supplemented with phytase A (Quantum Blue, manufactured by AB Vista Feed Ingredients, Marlborough, UK); 3) control supplemented with phytase B (Quantum XT, produced by AB Vista Feed Ingredients, Marlborough, UK); and 4) control supplemented with phytase C (Phyzyme TPT, manufactured by Danisco Animal Nutrition, Marlborough, UK). All the 3 phytase preparations were Escherichia coli–derived 6-phytase, and all applied at a rate of 1,000 FTU/kg of feed. Phytases A and C were supplemented as granular formulations, while phytase B was applied in liquid form. Each treatment included 6 replicates of 16 birds per replicate. Feed and water were offered to birds for
Table 1. Ingredient composition and calculated nutrient levels of basal diet. Ingredients4 Wheat Soybean meal Canola meal (solvent) Meat meal Canola oil Limestone Salt Sodium bicarbonate L-lysine HCl DL-methionine L-threonine Choline Cl 70% Mineral premix1 Vitamin premix2 Salinomycin (12%) Zn bacitracin (15%) Xylanase (Porzyme 93010, Dupont) Calculated nutrient level4 ME poultry (MJ/kg) Crude protein Calcium Phosphorus, NPP5 Phosphorus, total Sodium Choline (mg/kg) Dig3 lysine Dig methionine Dig methionine + cysteine Dig tryptophan Dig isoleucine Dig arginine Dig threonine Dig valine
Starter 60.3 24.2 9.6 1.7 1.8 1.1 0.31 0.20 0.27 0.21 0.03 0.11 0.075 0.05 0.05 0.033 0.005
Grower 59.1 25.0 10.0 1.0 3.1 0.9 0.21 0.20 0.10 0.16 0.00 0.04 0.075 0.05 0.05 0.033 0.005
12.35 12.56 23.1 22.5 0.75 0.60 0.25 0.20 0.54 0.52 0.22 0.18 1850 1470 1.20 1.10 0.44 0.48 0.84 0.80 0.19 0.27 0.78 0.85 1.26 1.29 0.74 0.70 0.92 0.95
1 Mineral mix supplied the following per kilogram of diet: Mn, 120 mg; Zn, 100 mg; Fe, 40 mg; Cu, 16 mg; Se, 0.3 mg; I, 1.25 mg. 2 Vitamins supplied the following amounts per kilogram of diet: vitamin A, 12,000 IU; cholecalciferol, 5,000 IU; vitamin E, 75 IU; vitamin K, 3 mg; vitamin B12 , 16 mcg; riboflavin, 8 mg; pantothenic acid, 13 mg; nicotinic acid, 55 mg; folic acid, 2 mg; biotin, 0.2 mg. 3 Dig = digestible. 4 Percent unless otherwise indicated. 5 Non-phytate phosphorus.
ad libitum consumption. The lighting program was 24 h of light during the first 3 d, 23 h of light from 4 d to 7 d, 20 h of light from 8 d to 10 d, and 18 h of light thereafter until the end of the study. Birds were fed in 2 phases: starter (0–21 d) and finisher (21–28 d). The basal diet was formulated to be deficient in ME, available P, and Ca. The ingredient composition of the basal diet and nutrient levels are shown in Table 1. A basal diet with the nutrient composition of the control was mixed first and then evenly divided into 4 groups, with the first group being the control, and the remaining 3 were treated with different phytases according to the experimental design. The starter diets were supplied in mash form, whereas the grower diets were pelleted. Pelleting temperature was maintained between 65 and 70◦ C. Pellets were spread onto a cool clean surface to a depth of 3 cm for cooling. After cooling, the liquid form, phytase B was applied as a diluted spray
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influence of phytase on dietary AME is inconsistent (Ravindran et al., 1999a; Shirley and Edwards, 2003; Pirgozliev et al., 2011). Since the AME system does not take into account the energy partitioned to production and heat increment, it has been suggested that the net energy (NE) system may offer an advantage in estimating energy utilization in poultry diets. Pirgozliev and Bedford (2013) found that dietary net energy for production (NEp) had a higher correlation to performance when compared to AME. This suggests that NE may be a more sensitive way to evaluate broiler response to phytase supplementation. Similar findings were also reported by Olukosi et al. (2008). Previous research focusing solely on the effect of phytase on AME may have underestimated the full value of exogenous phytase in the improvement of broiler performance. It was hypothesized that supplementation of phytase at an unconventionally high level (1,000 FTU/kg feed) improves broiler performance by limiting heat increment and producing a higher NE:ME ratio. Thus, the objective of the current study was to investigate the effects of 3 supplemental phytases, all used at a high dosage (1,000 FTU/kg), on growth performance, NE, relative digestive tract development, and bone mineralization in broiler chickens.
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WU ET AL. Table 2. The analyzed nutrients and phytase activity of diets. Treatment Starter
Grower
Control Control Control Control Control Control Control Control
(basal) + phytase + phytase + phytase (basal) + phytase + phytase + phytase
Calcium,%
P, total,%
Analyzed phytase activity in feed samples (FTU/kg)
0.84
0.55
0.63
0.54
74 823 794 1,072 467 1,552 1,480 1,649
A B C A B C
Sample Collection On d 21, 2 birds per pen were removed, weighed, and euthanized by cervical dislocation. The digestive tract, from the proventriculus to small intestine, was carefully excised. The weight of proventriculus plus gizzard was recorded. The weight of the small intestine (from the beginning of duodenum to the ileocecal junction) was obtained after the contents inside were emptied by gentle digital expression. The whole left leg of those 2 birds per pen was excised from the fresh carcass, sealed in plastic bags to minimize moisture loss, and then stored at –20◦ C until they were used for the determination of bone characteristics.
Net Energy Measurement At 22 d of age, 32 birds (8 birds from each treatment) were randomly selected and transferred from the rearing floor pens to 16 calorimeter chambers in a temperature-controlled room for a 3-d acclimatization. The birds were given their respective test diets, each with 4 replicates of 2 birds housed in a closed-circuit calorimeter chamber. The closed-circuit calorimeter chambers were newly constructed by the workshop of University of New England, and the design of the closed-circuit calorimetric chambers and the measurement of O2 consumptions and CO2 release by the birds using the chamber have been described previously (Swick et al., 2013). Briefly, the chamber was equipped with a wire-mesh cage, chamber air circulated through KOH to absorb CO2 and silica gel to remove moisture. An oxygen cylinder was fitted to replenish
O2 through pressure-controlled solenoid valve, and oxygen consumption was measured gravimetrically. The recovery of CO2 was analyzed by barium carbonate precipitation (Annison and White, 1961). Air-conditioning units were used maintain constant temperatures of 22 ± 1◦ C inside the room and 24 ± 1◦ C inside the chambers. Light was controlled to achieve 15 lux at the bird level. The heat production (HP) was estimated using the Brouwer equation (Brouwer, 1965): kcal total heat = 3.866 × L of oxygen consumed + 1.200 × L of CO2 expired. The AME was measured using the total collection method (Bourdillon et al., 1990). Excreta were collected and feed consumption recorded each day. The excreta from each chamber were thoroughly mixed into slurry using a blender and sub-samples taken for gross energy (GE) analysis. The experimental diets and excreta were analyzed R for GE using an adiabatic bomb calorimeter (IKA WERKE, C7000, GMBH and Co., Staufen, Germany). Benzoic acid was used as a standard. The respiratory quotient (RQ) of the 3-d run was calculated as the ratio of the volume of CO2 expired to the volume of O2 consumed. Heat increment (HI) was calculated by deducting fasting HP from total HP. The value of 450 kJ/kg BW0.70 per bird per day was applied as fasting HP, which corresponds to the asymptotic HP (at zero activity) during a 24-h fast, as proposed by (Noblet et al. 2010a, 2010b). NE was calculated as ME intake minus HI divided by consumption of feed on an as-is basis.
Tibia Ash, Toe Ash, and Tibia Bone Breaking Strength The legs of the 2 sacrificed birds were thawed, and tibia bones were severed and defleshed without boiling; the middle toe was clipped and cleaned of any waste material such as litter and excrement. Skin, flesh, and toenail of the middle toe were kept intact. The tibias and toes were dried at 105◦ C for 24 h and placed in a desiccator, and the weights were recorded. Tibia breaking strength was measured using an Instron instrument (Model 1011 Instron Universal Testing Machine, Instron Corp., Canton, MA). The instrument was
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(250× dilutions) of the received product. Each batch of pellets was sprayed evenly twice. The analyzed phytase activity and the inclusion level of Ca and total P of feed samples is shown in Table 2. One phytase unit is defined as the amount of enzyme required to release 1 μmol of inorganic P/min from sodium phytate at 37 ± 0.1◦ C and pH 5.5. Dietary phytase levels were analyzed by Pacific Lab Services (Singapore) according to method 2000.12 ( AOAC International, 2005). Dietary inclusion level of Ca and total P were analyzed using inductively coupled plasma optical emission spectrometer, and perchloric acid and hydrogen peroxide were used for digestion (Anderson and Henderson, 1986).
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MICROBIAL PHYTASE, NET ENERGY AND BONE CHARACTERISTICS Table 3. Responses of growth performance and relative organ weights from 0–21 d to 3 phytases1 . Treatment
BW g
Feed intake g
Feed/ gain g/g
Livability %
Gizzard + Proventriculus2
Small Intestine2
Control Control + phytase A Control + phytase B Control + phytase C SEM P>F
761b 853a 868a 859a 12.4 < 0.001
1,077b 1,223a 1,190a 1,164a 17.1 < 0.01
1.512 1.518 1.447 1.433 0.02 0.53
96.88 100 100 98.96 0.81 0.51
14.60 13.77 13.98 14.13 0.34 0.86
28.25a 24.11b 24.27b 26.68a,b 0.57 < 0.05
means in a column not sharing a common superscript differ (P < 0.05). The values are the means of 6 replicates from each treatment. 2 Relative weight (g/kg body weight). a,b 1
Statistical Analysis The data were analyzed using the SPSS 21.0 software program (SPSS Inc., Chicago, IL). One-way ANOVA was used to determine the difference among the treatments. If significant treatment effects were observed, the differences of means between treatments were determined using the least significant difference test. Differences were considered significant at P < 0.05.
RESULTS Table 1 shows expected and recovered phytase from experimental mixed feeds. Table 3 shows the results of the 21-d performance experiment. The supplementation of the 3 microbial phytases increased the average BW at 21 d (P < 0.001). The addition of phytases A, B, and C improved the average BW by 12.1, 14.1, and 12.9%, respectively, compared with the birds fed the control diet. A similar trend was observed for feed intake. Feed intake of birds fed the diets supplemented with all 3 phytases was significantly increased by 13.6, 10.5, and 8.1%, respectively (P < 0.01). None of the phytases had any effect on FCR (P >0.05). Phytase had no impact on livability (P > 0.05). Supplementation of phytases A and B reduced the relative weight of the small intestine compared to the control by 14.7 and 14.1%, respectively (P < 0.05). Results shown in Table 4 indicate that during the 3-d period (d 25–27) of measurements of respiratory gases in chambers, no difference in RQ, performance, HP, or NE:ME ratio was observed between treatments (P > 0.05). NE per kg of diet was significantly increased by phytases A and B (P < 0.05) by 5.6 and 5.1%, respectively. It was also observed that phytase B tended to improve MEI (P = 0.07), and both phytases A and B tended to improve ME in comparison with phytase C (P = 0.09).
Phytase supplementation increased the relative weights of tibia ash and toe ash (Table 5), with phytase B improving the relative weight of tibia ash by 11.0% (P < 0.01) and phytase A numerically (P = 0.097) increasing the relative weight of tibia ash by 6.2%. Similarly, both phytases A and B resulted in, respectively, 18.3 and 17.6% higher relative weight of toe ash compared to the control (P < 0.001). However, phytase supplementation had no effect (P > 0.05) on tibia bone breaking strength.
DISCUSSION The study used diets formulated to be deficient in available P, Ca, and ME to examine if 3 different E. coliderived phytases elicited energy responses differently. Phytase recovery from diets is shown in Table 1. The results suggest reasonable expected recovery. Differences in assayed phytase levels in the non-supplemented starter and grower control basal diets were observed, and supplemented starter diets were more variable than grower diets. The starter diets had higher limestone, salt, and meat meal inclusion than the grower diets. It is possible that these minerals may have reduced the activity of endogenous phytase from wheat in the starterdiet assay. Feeding study results indicated that supplementation with intrinsically thermostable phytases A and B did not support the hypothesis of improving performance through reduced HI of diets. Although NE was increased in diets supplemented with phytase A and B, no effect of these 2 phytases was observed on HI, which demonstrated that improved performance mainly originated from increased ME concentration in the diets induced by phytases A and B. When energy responses to phytase supplementation are measured using the AME system, the results are often equivocal (Tejedor et al., 2001; Shirley and Edwards, 2003; Cowieson and Adeola, 2005; Chung et al., 2013). But the net-energy system has been considered a more refined way of estimating energy partitioning and utilization in animals, as this system takes into account total HP and HI due to digestion and metabolism of feed (Swick et al., 2013). The current study found that the dietary inclusion of the 2 intrinsically thermostable phytases increased dietary NE, which was in agreement
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equipped with 50-kg-load cell with a crosshead speed of 50 mm/min. Tibias were supported on a 3.35-cm span (Shafer et al., 2001). Moisture-free tibia ash and toe ash were determined by ashing in ceramic crucibles for 24 h at 615◦ C. Tibia ash and toe ash were expressed as the percentages of tibia ash and toe ash relative to tibia and toe dry weights.
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WU ET AL. Table 4. Energy partition and performance responses to 3 different phytases in broilers during 25 and 28 d of age1 . Control no phytase
Phytase A
Phytase B
Phytase C
SEM
P>F
1,402 88.0 1.595 1,368 851 401 1.02 13.87 9.80b 70.7
1,404 92.8 1.560 1,460 851 401 1.04 14.26 10.35a 72.6
1,391 97.9 1.510 1,503 866 416 1.02 14.23 10.30a 72.3
1,386 86.4 1.618 1,354 825 375 1.02 13.65 9.86b 72.3
13.9 2.4 0.023 23.7 7.2 7.2 0.004 0.103 0.088 0.397
0.972 0.407 0.456 0.068 0.267 0.267 0.354 0.086 0.024 0.288
BW, g ADG, g Feed conversion ratio, g/g MEI, kJ/kg BW0.70 HP, kJ/kg BW0.70 HI, kJ/kg BW0.7 RQ ME, kJ/g feed DM NE, kJ/g feed DM NE:ME (%)
means in a row not sharing a common superscript differ (P < 0.05). The values are the means of 4 replicates from each treatment.
a,b 1
Table 5. Bone mineralization changes in response to 3 phytases in broilers during 0–21 d of age1 .
Control Control + phytase A Control + phytase B Control + phytase C SEM P>F
Tibia ash (%) b
35.6 37.8a,b 39.5a 37.2a,b 0.5 0.043
Toe ash (%) b
11.1 13.1a 13.1a 12.0b 0.2 0.001
Bone breaking strength (N) 82.2 72.8 90.5 66.2 3.5 0.055
means in a column not sharing a common superscript differ (P < 0.05). The values are the means of 6 replicates from each treatment.
a,b 1
with previous reports (Olukosi et al., 2008; Pirgozliev and Bedford, 2013). However, the improvement of dietary NE largely originated from the increased ME of the diets supplemented with phytase A and phytase B. This contradicts the hypothesis of lower HP and HI as a result of phytase supplementation. Furthermore, there was no effect of phytase on the ratio of NE to ME. In the current study, a tendency for phytase supplementation to improve ME was observed in the following order: A > B > control = C (P < 0.09). This energy effect of phytase is likely due to improved availability of energy-yielding components in the diet and perhaps improved amino acid digestibility. Phytase has been shown to denature Ca-phytate complexes, which are involved in the formation in the gut lumen of metallic soaps that limit energy utilization from fat (Ravindran et al., 2001). There is much evidence that exogenous phytase is capable of increasing amino acid digestibility from a variety of diet types (Namkung and Leeson, 1999; Camden et al., 2001; Ravindran et al., 2001). Phytate is an integral component of the cell-wall matrix in wheat (Frolich, 1990), and supplementation with phytase may increase the accessibility of digestive enzymes to their respective substrates, thus increasing the ME value of diets. Phytase may also reduce endogenous losses, thereby increasing ME. There is large variation in endogenous losses among birds fed different feed ingredients (Siriwan et al., 1993; Ravindran et al., 2006). Feed intake, enzyme supplementation, and other feed additives can have an effect on endogenous losses (Angkanaporn
et al., 1994; Cowieson et al., 2006; Pirgozliev et al., 2008). Ravindran et al. (2006) found phytate increased Na losses by 60% in ad libitum-fed broiler chickens, while phytase reduced these losses by 66%. Similar effects of phytase were reported on sialic acid secretion by Cowieson et al. (2004) and Pirgozliev et al. (2007). The reduction in endogenous losses would be expected to lead to a reduction of the energy required for maintenance, thus allowing a greater proportion of metabolizable energy to be partitioned towards growth. Nevertheless, it was unexpected that the coated phytase failed to deliver the same result on dietary ME and NE as the 2 intrinsically thermostable counterparts. The discrepancy on effectiveness may be related to the rate of reaction of phytate degradation. The mean retention time for digesta within the gastrointestinal tract is less than 3.5 h (Hughes, 2008), and only a small portion of that time is spent in the crop, gizzard, and proventriculus, where phytate is soluble and available for dephosphorylation and reaction with phytase. In this regard, it is possible that the intrinsically thermostable phytases, without coating, are more soluble in the upper gastrointestinal tract and are more bioactive in this region than coated phytase. The use and selection of phytase product type would, of course, depend on whether it is intended for pre- or post- pellet application. Dietary phytate has been considered as an antinutrient as it forms complexes with minerals, starch, and protein, rendering them less available for monogastric animals (Sebastian et al., 1996; Ravindran et al., 1999b; Shelton and Southern, 2006). Monogastric
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Treatment
MICROBIAL PHYTASE, NET ENERGY AND BONE CHARACTERISTICS
especially protein and lipids, so there is a chance that the fixed fasting HP did not represent the true fasting HP in birds fed diets with phytase. Therefore, it is recommended to measure fasting HP directly in future trials when exogenous enzymes are treated. It is concluded that supplemental microbial phytase can beneficially influence growth performance and bone mineralization in ME-, Ca-, and available P-deficient, wheat-based diets. This study has shown that while dietary inclusion of intrinsically thermostable phytases at dose levels of 1,000 FTU/kg feed have the capacity to improve dietary ME and NE. This was a result of increased MEI and was not caused by reduced HI. None of the examined phytases lowered HP or HI, and none increased the ratio of NE to ME.
REFERENCES Anderson, D. L., and L. J. Henderson. 1986. Sealed chamber digestion for plant nutrient analysis. Agron. J. 78:937–938. Angkanaporn, K., M. Choct, W. L. Bryden, E. F. Annison, and G. Annison. 1994. Effects of wheat pentosans on endogenous aminoacid losses in chickens. J. Sci. Food Agric. 66:399–404. Annison, E. F., and R. R. White. 1961. Glucose utilization in sheep. Biochem. J. 80:162–169. AOAC International. 2005. Official methods of analysis. 18th ed., AOAC International, Arlington, VA. Applegate, T. J., R. Angel, and H. L. Classen. 2003. Effect of dietary calcium, 25-hydroxycholecalciferol, or bird strain on small intestinal phytase activity in broiler chickens. Poult. Sci. 82:1140–1148. Bourdillon, A., B. Carre, L. Conan, M. Francesch, M. Fuentes, G. Huyghebaert, W. Janssen, B. Leclercq, M. Lessire, J. McNab, M. Rigoni, and J. Wiseman. 1990. European reference method of invivo determination of metabolizable energy in poultry: reproducibility, effect of age, comparison with predicted values. Br. Poult. Sci. 31:567–576. Brouwer, E. 1965. Report of subcommittee on constants and factors. Proc. the 3rd symposium on energy metabolism, European Federation of Animal Science Publication 11. (Ed. KL Blaxter). 441–443. Camden, B. J., P. C. H. Morel, D. V. Thomas, V. Ravindran, and M.R. Bedford. 2001. Effectiveness of exogenous microbial phytase in improving the bioavailabilities of phosphorus and other nutrients in maize-soya-bean meal diets for broilers. Anim. Sci. 73:289–297. Chung, T. K., S. M. Rutherfurd, D. V. Thomas, and P. J. Moughan. 2013. Effect of two microbial phytases on mineral availability and retention and bone mineral density in low-phosphorus diets for broilers. Br. Poult. Sci. 54:362–373. 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., and O. Adeola. 2005. Carbohydrases, protease, and phytase have an additive beneficial effect in nutritionally marginal diets for broiler chicks. Poult. Sci. 84:1860–1867. Cowieson, A. J., M. Hruby, and E. E. M. Pierson. 2006. Evolving enzyme technology: impact on commercial poultry nutrition. Nutr. Res. Rev. 19:90–103. Cowieson, A. J., P. H. Selle, and V. Ravindran. 2008. Influence of dietary phytic acid and source of microbial phytase on ileal endogenous amino acid flows in broiler chickens. Poult. Sci. 87:64– 64. Cowieson, A. J., P. Wilcock, and M. R. Bedford. 2011. Super-dosing effects of phytase in poultry and other monogastrics. Worlds Poult. Sci. J. 67:225–235. Esteve-Garcia, E., J. Brufau, A. Perez-Vendrell, A. Miquel, and K. Duven. 1997. Bioefficacy of enzyme preparations containing betaglucanase and xylanase activities in broiler diets based on barley or wheat, in combination with flavomycin. Poult. Sci. 76:1728– 1737.
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animals do not produce enough endogenous phytase (Maenz and Classen, 1998; Applegate et al., 2003) and, as a result, exogenous phytase is required in the diets to alleviate the detrimental effects of phytate. In the current study, the phytase-supplemented groups all had substantially higher average BW and feed intake compared with the control, but had little impact on FCR. P deficiency in the diet generally compromises broilers’ appetite, lowers the blood P level, decreases the circulating levels of growth hormone (Parmer et al., 1987) and consequently reduces the rate of weight gain. It has been reported that phytase-improved feed intake in the birds fed available P-deficient diets, and thus weight gain was increased but feed efficiency did not show improvement (Shaw et al., 2010; Karimi et al., 2013). The results of this experiment agree with this mode of action of phytase. The data in the present study showed little impact of phytase addition on HP or HI. However, birds had reduced small intestine size in the treatments supplemented with phytases A and B. According to Spratt et al. (1990), although the liver and the gastrointestinal tract account for approximately 3% of the bodyweight of a hen, they may consume up to 26% of the fasting HP, and the total cost of maintenance may take up to 75% of total fasting HP. Previous studies (EsteveGarcia et al., 1997; Wu et al., 2004) showed that the use of exogenous enzymes often reduced the size of the energetically demanding organs, such as the pancreas and the gut. In the present study, the reduced small intestine size observed in the groups supplemented with phytases A and B is in accord with the earlier findings. However, phytase addition in this study did not have any impact on heat increment or HP. This was unexpected, as it was hypothesized that less energy would be consumed by a smaller gastrointestinal tract and result in lower HP. The likely explanation of the lack of effect on HP in the phytase-supplemented groups is that the enzyme increased the energy need for maintenance of skeletal muscles as well as the energy expenditure associated with the absorption of some energydependent active transported nutrients and minerals (Kies et al., 2005). Considering some phytase addition has increased the relative weight of tibia ash (phytase B, by 11.0%) and toe ash (phytase A, by 18.3%; phytase B, by 17.6%) in the present study, it may be postulated that a higher amount of heat was generated while more P or Ca was released, absorbed, and utilized in the phytase-supplemented treatments. Moreover, a fixed figure for fasting HP (450 kJ/kg BW0.70 per bird per day) was applied in the current study, based on the findings of (Noblet et al. 2010a, 2010b), which suggests that fasting HP in 0.5 to 3.0 kg broilers is proportional to BW0.70 with values ranging between 420 and 450 kJ/kg BW0.70 /d. However, from a practical point of view, the fasting HP figure may be fully justified in broilers fed diets with similar chemical characteristics, but phytase supplementation may alter fasting HP by changing the way broilers digest and utilize nutrients,
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