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ANIMAL NUTRITION |
* Department of Animal Sciences, University of Illinois, Urbana 61801 and
and
United Feeds, Inc., Sheridan, IN 46069
Abstract
A pig trial and a chick trial were done to determine the effect of high levels of Zn and Cu on the P-releasing efficacy of phytase. Ninety-nine individually fed pigs (7.2 kg) were given ad libitum access to one of 11 experimental diets for a period of 21 d. Fibula ash (mg) was regressed against supplemental inorganic P (iP) intake (g) to establish the standard curve, from which phytase treatments were compared to determine P-releasing efficacy. The basal diet was a corn-soybean meal diet with no supplemental P (21% CP, 0.075% estimated available P, 130 mg of Zn/kg, as-fed basis). Diets included three graded levels of supplemental iP (0, 0.075, 0.150%) from reagent-grade KH2PO4, two levels of phytase (500 and 1,000 FTU/kg) from EcoPhos, 1,500 mg of Zn/kg from either Waelz ZnO or basic Zn chloride (Zn5Cl2(OH)8), and all combinations of phytase and Zn. One phytase unit (FTU) was defined as the amount of enzyme required to release 1 µmol of iP per minute from sodium phytate at 37°C and pH 5.5. Phytase supplementation improved (P < 0.01) weight gain, G:F, and fibula ash (% and mg). Bone ash (mg) was highest (P < 0.01) for pigs fed diets containing 1,000 FTU/kg of phytase. Supplemental Zn had no effect (P > 0.50) on growth performance, but decreased (P < 0.05) fibula ash (mg). Comparison of the phytase treatments to the standard curve (r2 = 0.87) revealed P-release values of 0.130 and 0.195% for 500 and 1,000 FTU of phytase/kg, respectively, in the absence of Zn, whereas in the presence of Zn (pooled), P-release values were decreased (P < 0.01) to 0.092 and 0.132%, respectively. The effects of high levels of supplemental Zn (basic Zn chloride) and Cu (CuSO4•5H2O) on phytase efficacy also were investigated in a 12-d chick trial. Dietary treatments were arranged according to a 23 factorial, with two levels each of supplemental phytase (0 and 500 FTU/kg from EcoPhos), Zn (0 and 800 mg/kg), and Cu (0 and 200 mg/kg). There was a phytase x Zn interaction (P < 0.01) for tibia ash. Thus, Zn supplementation decreased tibia ash in the presence, but not in the absence, of phytase. Supplemental Cu did not affect (P > 0.30) the response to phytase. These results suggest that pharmacological levels of Zn chelate the phytate complex, thereby decreasing its availability for hydrolysis by phytase.
Key Words: Chicks • Copper • Phytase • Pigs • Zinc
Introduction
Zinc is routinely fed at pharmacological doses (1,500 to 3,000 mg/kg) to newly weaned piglets for the possible alleviation or prevention of diarrhea (Holm, 1988
; Poulsen, 1995) and for growth promotion (Hahn and Baker, 1993; Mavromichalis et al., 2001; Case and Carlson, 2002). Recent in vitro data, however, showed that Zn2+ was a potent inhibitor of phytase-mediated phytate-P hydrolysis, which the authors postulated was due in part to Zn binding causing a conformational change in the phytate moiety, thereby rendering it less accessible to phytase (Maenz et al., 1999). Zinc has been shown to form a complex with phytic acid in vitro that is quite stable and precipitated out of solution at Zn:phytate molar ratios of 3.5 to 4:1 (Champagne and Fisher, 1990). Additionally, it appeared that one Zn2+ ion might bridge two phytate molecules over time (Champagne and Fisher, 1990).
Supplemental Cu is also extensively used for growth promotion and antimicrobial action in both pigs and poultry (Edmonds and Baker, 1986; Cromwell et al., 1998; Ewing et al., 1998). Similar to Zn, Cu was shown to form a complex with phytate, but the Cu-phytate complex was soluble at all pH values (Champagne and Fisher, 1990). Due to the extensive use of pharmacological levels of both of these minerals in nonruminant nutrition, and their interactions with phytate, our objective was to determine their effects, alone and in combination, on the P-releasing efficacy of phytase in young pigs and chickens.
Materials and Methods
Phytase
EcoPhos (Phytex LLC, Portland, ME) is a phytase that was cloned from Escherichia coli and expressed in a yeast-expression system as described by Rodriguez et al. (1999a,b; 2000). Classified as a 6-phytase, it is a recombinant enzyme produced from the appA2 gene isolated from E. coli found in pig intestine (Rodriguez et al., 1999a). EcoPhos exhibits optimal activity at pH 2.5 to 3.5 (Rodriguez et al., 1999a). A similar E. coli-derived phytase was shown by Rodriguez et al. (1999b) to be more resistant to the gastric enzyme pepsin than Natuphos (BASF, Mt. Olive, NJ). Previously, our laboratory reported that 400 FTU of EcoPhos/kg released 0.11% P in young pigs, whereas 500 FTU/kg released an average of 0.13% P in chicks fed P-deficient corn-soybean meal (SBM) diets (Augspurger et al., 2003). The enzyme was assayed for phytase activity before inclusion in experimental diets as described previously by Han et al. (1999). One phytase unit (FTU) was defined as the amount of enzyme required to release 1 µmol of inorganic P (iP) per minute from sodium phytate at 37°C and pH 5.5 (Augspurger et al., 2003).
Pig Trial
The University of Illinois Institutional Animal Care and Use Committee approved all experimental procedures. The pig trial was conducted at the United Feeds Burton-Russell Research Farm (Frankfort, IN) with 99 AusGene barrows (7.2 kg). The pigs were weaned at approximately 15 d of age and housed in an environmentally controlled nursery with ad libitum access to a corn-SBM-whey-plasma starter diet adequate in all essential nutrients (NRC, 1998). At 1 wk postweaning, pigs were moved to the experimental building equipped with individual pens (0.5 m x 0.9 m) and allowed 4 d to acclimate to the facility. Pigs were then deprived of feed for 12 h to minimize gut fill, weighed, assigned to nine uniform blocks based on BW and location in barn, and then allotted randomly to treatment diets from within blocks. Pigs were penned individually and given ad libitum access to the experimental diets for a period of 21 d, during which time pigs and feeders were weighed weekly. Nine pigs received each of 11 experimental diets.
The basal diet (Table 1) was formulated to be deficient in P, containing an analyzed level of 0.32% total P. Using average P bioavailability estimates of 25% for SBM and 15% for corn (Cromwell, 1992), the basal diet contained an estimated 0.075% available P. With the exception of P, the diet was formulated to be adequate to super adequate in all other nutrients, including Ca (0.70%), Zn (130 mg/kg), and cholecalciferol (16.5 µg/kg) for 10- to 20-kg pigs (NRC, 1998). No supplemental Cu, other than that included in the trace-mineral salt (8 mg/kg), was present in the diet.
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The pig trial was designed to measure the effects of pharmacological levels of Zn in the absence and presence of supplemental phytase on fibula ash and bioavailable P release in young pigs fed P-deficient corn-SBM diets (Table 1
). There were 11 experimental diets, including the basal diet, the basal diet supplemented with two levels of iP from KH2PO4 (0.075 and 0.150%), two levels of phytase from EcoPhos (500 and 1,000 FTU/kg), two sources of Zn (1,500 mg/kg from basic Zn chloride [ZC, Zn5Cl2(OH)8, Micronutrients, Inc., Indianapolis, IN] or Waelz-processed ZnO [Southeastern Minerals, Bainbridge, GA]), and the four combinations of phytase and Zn additions. The basal diet and the two levels of iP were used to construct the standard curve. Zinc supplemented at a level of 1,500 mg/kg from ZC was previously found to exhibit growth-promoting efficacy in young pigs similar to that obtained with 3,000 mg of Zn/kg from Waelz-processed ZnO (Mavromichalis et al., 2001).
Chick Trial
The University of Illinois Institutional Animal Care and Use Committee approved all experimental procedures. The trial employed New Hampshire x Columbian Plymouth Rock male chicks housed in thermostatically controlled starter batteries with raised wire floors in an environmentally controlled building that provided 24 h of light. Both feed and water were provided for ad libitum consumption. During the first 7-d after hatching, chicks were fed a corn-SBM diet adequate in all essential nutrients (NRC, 1994). After a period of overnight feed withdrawal, all chicks appearing normal and healthy were weighed, and those chicks within a narrow weight range were selected. The selected chicks were then wing-banded and randomly assigned to pens, which were in turn randomly assigned to dietary treatments. Five replicate pens of four chicks per pen were allowed to consume each experimental diet from d 8 to d 20 after hatching.
The basal diet (Table 1) was a corn-SBM diet, designed to be deficient in P but superadequate in cholecalciferol (i.e., 25 µg/kg diet). An additional 0.05% iP from reagent-grade KH2PO4 was added to increase the available P level to 0.15% in an effort to counteract the potential negative effect of supplemental Zn and Cu on bone ash. The basal diet was analyzed to contain 0.42% total P. Calcium in the basal diet was set at 0.75%, a level lower than the NRC (1994) requirement of 1.00%, in an effort to control Ca:P imbalance and thereby allow maximum responses to phytase. The basal diet contained both Zn (107 mg/kg) and Cu (18 mg/kg) at levels in excess of the NRC (1994) requirement. In all cases, experimental diets were first limiting in bioavailable P (Biehl and Baker, 1997a,b).
At the termination of the experiment, BW of individual chicks and pen feed intakes were measured for calculation of weight gain, feed intake, and G:F. Chicks were then killed by CO2 inhalation for collection of right tibiae for bone ash determination. Tibiae were pooled by replicate pens and processed as previously described for the pig bones.
The objective of the chick trial was to determine the effect of high levels of Zn and Cu, alone or in combination, on the efficacy of phytase in chicks fed P-deficient corn-SBM diets. Dietary treatments were arranged in a 23 factorial, with factors being phytase (0 and 500 FTU/kg from EcoPhos), Zn (0 and 800 mg/kg from ZC), and Cu (0 and 200 mg/kg from feed-grade CuSO4•5H2O). These levels of Zn and Cu were previously found not to depress growth in chicks fed fully fortified corn-BM diets (Funk and Baker, 1991; Persia, 2003).
Diet Analysis
Crude protein in all basal diets was determined in triplicate (AOAC, 1995). Quadruplicate samples of basal diets from each of the trials were dry ashed as described for the bones and then wet ashed for quantification of total P (Augspurger et al., 2003).
Statistical Analysis
For the pig trial, analysis of variance was performed on individual pig data as a randomized complete-block design. For the chick trial, ANOVA was performed on pen means data as a completely randomized design. Treatment means within each trial were compared using orthogonal single-df comparisons. In the pig trial, fibula ash (mg) was regressed on supplemental iP intake (g) to construct the standard curve. Using bone ash responses to supplemental phytase, the regression equation was solved for bioavailable P intake (g) and the solution was divided by feed intake and multiplied by 100 to yield bioavailable P release. Also, multiple linear regression of fibula ash as a function of supplemental phytase intake was done to compare bone ash responses per unit of phytase intake (i.e., slope values) in pigs fed diets with and without 1,500 mg/kg of added Zn.
Results
Pig Trial
Weight gain, G:F, and fibula ash (mg) increased linearly (P < 0.01) in response to iP supplementation (Table 2). Phytase supplementation resulted in increases (P < 0.01) in all response criteria, with a dose effect (P < 0.01) for weight gain, G:F, and fibula ash (mg). Zinc supplementation (1,500 mg/kg) resulted in depressed (P < 0.05) fibula ash (mg) values, with a slight difference (P < 0.10) between the Zn sources for fibula ash concentration. Regression of fibula ash (mg) on supplemental iP intake (g) resulted in an excellent fit for the standard curve regression equation (Y = 430.5 ± 16.8 + 10.2 ± 1.1X; r2 = 0.87). Phytase supplementation at 500 and 1,000 FTU/kg resulted in bioavailable P release values (%) of 0.130 and 0.195% (± 0.019), respectively. Zinc supplementation (pooled) at these two phytase levels reduced (P < 0.01) efficacy values to 0.092 and 0.132% (± 0.013), respectively.
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). Zinc supplementation resulted in depressed tibia ash values (mg and %) in the presence of phytase, but not the absence (phytase x Zn interaction; P < 0.01). Copper supplementation resulted in small, but significant (P < 0.05) increases in bone ash values.
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The results herein are the first to show a quantitative effect of Zn supplementation on the in vivo efficacy of phytase for improving bone ash. In the young pig, dietary Zn supplementation at 1,500 mg/kg (regardless of source) resulted in a 30% decrease in the P-releasing efficacy of phytase. Similarly, in the chick, dietary Zn supplementation (800 mg/kg) reduced tibia ash by 14% in the phytase-supplemented diets for chicks. This verifies the in vitro work of Maenz et al. (1999) that showed a potent inhibitory effect of Zn2+ on phytate hydrolysis by phytase. At pH 7, Zn2+ at a molar ratio to phytate of 0.35:1 inhibited phytate-P hydrolysis by 50%, whereas at pH 4, the ratio was 9.9:1. Assuming all phytate-P in our pig basal diet (approximately 0.24%) was in the hexaphosphate form of phytate (Boland et al., 1975; Raboy et al., 2000), the total Zn2+ (basal plus supplemented):phytate molar ratio in our Zn-supplemented pig diets was approximately 2:1.
One could question the effect that Zn present in the basal diet might have had on phytase efficacy. Zinc in the basal pig diet (130 mg/kg) was provided at a surfeit level compared with the NRC (1998) requirement of 80 mg/kg for the 10- to 20-kg pig. In fact, a majority of the Zn in the basal diet (100 mg/kg) was provided by the trace-mineralized salt in the form of ZnO. In light of the surfeit amount of Zn already present, there is a possibility that the effect of supplemental Zn in our experiment was not maximized. Certainly the effect of supplemental Zn above the pig’s requirement on the efficacy of phytase is an area worthy of further research.
At first inspection of the fibula ash data, the effect of Zn supplementation on phytase efficacy does not appear to be great. In fact, 1,500 mg of Zn/kg addition to the phytase-containing diets for pigs resulted in a modest 9.5% decrease in fibula ash (Figure 2), but a 29% decrease in fibula ash per FTU of phytase intake (Table 2). The much more dramatic effect on phytase efficacy (% P release) cannot be explained by differences in feed intake (data not shown), as Zn supplementation did not result in any change in feed intake. The explanation can be found in an examination of the relationship between supplemental iP intake (g) and fibula ash (mg) accumulation (Figure 3). The slope of the standard-curve regression equation shows that a large change in supplemental iP intake resulted in only a small change in bone ash. Hence, the 9.5% decrease in fibula ash in response to Zn supplementation was the result of a 28% reduction in the calculated (available) P intake of pigs that received the phytase-supplemented diets. Hence, the reason for the difference in the magnitude of response between bone ash and phytase efficacy resulted from the differences in the magnitude of change between bone ash and available P intake.
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found through 31P NMR spectrum analysis that Zn2+ preferentially bound the 5-position phosphate of a specific phytate conformer, a type of binding that the authors theorized may allow the Zn2+ ion to bridge two phytate molecules. This led Maenz et al. (1999) to hypothesize that this proposed bridging might convert the phytate molecule to a conformation that is inaccessible to phytase-mediated hydrolysis. In vitro work with pure phytate (myo-inositol hexaphosphate) has shown that at pH 7, Zn phytate complexes will precipitate out of solution at a molar ratio of 4:1 within 3 h of incubation (Champagne and Fisher, 1990). The extent of precipitation per se as a cause of reduced phytase accessibility is unclear, as the Zn:phytate molar ratio in our diet was not that high (i.e., the dietary molar ratio of Zn:phytate was approximately 2:1), and the pH at which the in vitro experiment was run (7.0) is much higher than the pH of the pig stomach (approximately 3 to 4) (Yi and Kornegay, 1996; Radcliffe et al., 1998) This model also does not take into account the effect of additional cations that are present in a mixed diet that may be bound to the phytate molecule in the gut. In addition to Zn, phytate can bind Ca, Mn, Fe, and Mg (Erdman, 1979). High Ca, for example, enhanced the negative effect of phytate on zinc in both in vitro and in vivo settings (Byrd and Matrone, 1965; Morris and Ellis, 1980).
The Zn effect on the P-releasing efficacy of phytase could have significant implications in diets for early-postweaned pigs. Zinc is commonly supplemented to young pig diets for growth promotion (Hahn and Baker, 1993; Hill et al., 2001; Mavromichalis et al., 2001) and/or alleviation or prevention of diarrhea (Holm, 1988; Poulsen, 1995). These diets are typically complex starter diets containing a significant amount of milk products and plasma proteins, resulting in a small need for P supplementation. Nonetheless, these diets contain greater than 0.14% phytate-P (Hahn and Baker, 1993; Hill et al., 2000; 2001), and depending on the dietary formulation, they require some iP supplementation to meet the pig’s requirement for available P. Exogenous phytase, however, could be used to replace iP supplementation in this situation.
Copper (200 mg/kg) did not affect the P-releasing efficacy of phytase, measured as tibia ash, in our chick trial. This is interesting in light of data showing that phytic acid may bind more Cu2+ ions than Zn2+ ions at similar metal ion:phytate molar ratios and pH values (Champagne and Hinojosa, 1987). The Cu-phytate complex was soluble in solution, whereas the Zn phytate complex was insoluble and precipitated out of solution (Champagne and Fisher, 1990). Ondracek et al. (2002) reported no effect of Cu supplementation below 250 mg/kg, but 250 mg of Cu/kg decreased apparent P retention by 19% in chicks fed diets containing phytase. Copper is routinely supplemented at levels of 100 to 250 mg/kg as a growth promoter or antimicrobial agent in pigs (Edmonds and Baker, 1986; Cromwell et al., 1998) and chicks (Bakalli et al., 1995; Ewing et al., 1998). Because these diets that contain high levels of Cu are usually also high-phytate-containing diets, more research is needed to adequately determine the effect of these levels of Cu on the efficacy of phytase.
Implications
Pharmacological levels of zinc significantly decreased the efficacy of phytase for improving bone ash in both the young pig and chick. Although the potential utilization of phytase in early post-weaned pigs may be limited, this nevertheless shows that its inclusion level must be adjusted for zinc supplementation in the diet. Supplemental copper at 200 mg/kg did not affect phytase efficacy in chicks, but its effect at higher dose levels and in combination with higher levels of zinc is an important area of future research.
Footnotes
1 Funding for this research was provided in part by the State of Illinois thorugh the Illinois Council on Food and Agricultural Research (C-FAR). 
2 The authors acknowledge assistance of E. Parr, United Feeds, Inc., for help in diet mixing and daily management of the pig trial.
3 Present address: United Feeds, Inc., Sheridan, IN 46069.
4 Correspondence: 290 Animal Sciences Laboratory, 1207 W. Gregory Dr. (phone: 217-333-0243; fax: 217-333-7861; e-mail: dhbaker{at}uiuc.edu).
Received for publication September 9, 2003. Accepted for publication February 19, 2004.
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