Phytase-Induced Changes in Mineral Utilization in Zinc-Supplemented Diets for Pigs 1,2,3

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1 Published December 11, 2014 Phytase-Induced Changes in Mineral Utilization in Zinc-Supplemented Diets for Pigs 1,2,3 O. Adeola 4, B. V. Lawrence, A. L. Sutton, and T. R. Cline Department of Animal Sciences, Purdue University, West Lafayette, IN ABSTRACT: Forty-eight pigs (barrows:gilts, 1:1) with an average initial weight of 9.4 kg were used in a 2 2 factorial experiment to determine the influence of dietary phytase (0 or 1,500 phytase units/kg) and zinc ( 0 or 100 mg/kg) supplementation of a cornsoybean meal diet on the utilization of P, Ca, Cu, Mg, Mn, and Zn. After a 21-d growth experiment, feed was withheld for 24 h and blood was collected from the anterior vena cava of all pigs for plasma mineral analyses. Twenty-four barrows from the growth experiment were then placed in metabolism cages and used in an 8-d mineral balance study. All pigs were maintained on their previous diet. Growth rate was fastest ( P <.05) and feed efficiency was highest ( P <.05) for pigs fed phytase-supplemented diets. Feed intake was unaffected ( P >.05) by dietary treatment. Plasma P ( P <.01) and Mg (P <.05) concentrations increased with phytase addition. Plasma Zn concentration increased ( P <.05) when phytase was added to the diet containing no supplemental Zn, but plasma Zn concentration was not affected ( P >.05) by phytase when the diet was supplemented with 100 mg of Zn/kg. Apparent Ca, P, and Cu balance were improved ( P <.05) with phytase addition; however, Cu balance was reduced ( P <.05) by Zn supplementation. Zinc balance was increased ( P <.05) with supplemental zinc and phytase in the diet. These results indicate that the growth-promoting effect of phytase may be due to an overall increase in the availability of minerals. Key Words: Pigs, Phytase, Zinc, Minerals J. Anim. Sci : Introduction Phytate phosphorus, which is largely unavailable to nonruminant animals, accounts for approximately 60 to 80% of the total P in most grains (O Dell and Savage, 1960; Oberleas et al., 1973). Phytic acid forms complexes with other minerals, also reducing their availability (Davies and Nightingale, 1975; Nwokolo and Bragg, 1977; Brink et al., 1991, 1992; Pallauf et al., 1994ab). Phytate reportedly has the highest binding affinity for Cu and Zn (Maddaih et al., 1964; Vohra et al., 1965; Davies and Olpin, 1979). Evidence indicates that the binding of Zn to the phytate 1 Journal article no of the Purdue Univ. Agric. Res. Programs. 2 This research was partially funded by the Indiana Pork Producers Association, Indianapolis and Purdue Univ. CROSS- ROADS initiative. 3 The authors are grateful to BASF Corp., Parsippany, NJ for supplying the phytase, the staff of Purdue Swine Res. Unit for animal care, and Ray A. Sweet for technical assistance. 4 To whom correspondence should be addressed. Received June 29, Accepted July 14, molecule may be sufficient to decrease growth rate and Zn utilization in the absence of Zn supplementation (O Dell and Savage, 1960; Oberleas et al., 1962; Davies and Nightingale, 1975), and dephytinization of soy formula has been shown to increase Zn availability (Lonnerdahl et al., 1988). The use of phytase, an enzyme preparation that results in the stepwise removal of P from the phytate molecule (Maga, 1982), was reported (Nelson et al., 1971; Nasi, 1990) to increase P availability and growth rates in diets for chicks and pigs. These results have been validated by Simons et al. (1990) and Perney et al. (1993) for poultry and by Jongbloed et al. (1992), Cromwell et al. (1993), Lei et al. (1993a,b,c), and Mroz et al. (1994) for pigs. Phytase also has been shown to increase the availability and retention of Ca (Nasi, 1990; Simons et al., 1990; Young et al., 1993) and improve the absorption of Mg, Cu, Fe, and Zn (Nasi, 1990; Pallauf et al., 1992; Lei et al., 1993a). The objective of this experiment was to determine the influence of dietary phytase on the utilization of Ca, P, Zn, Mn, Mg, and Cu in diets supplemented with 0 or 100 mg of Zn per kilogram. 3384

2 PHYTASE- AND ZINC-SUPPLEMENTED DIETS FOR PIGS 3385 Table 1. Composition of diets (as-fed basis) Supplemental Zn, mg/kg: Item Phytase, PU/kg a : 0 1, ,500 Ingredient, g/kg Corn (7% CP) Soybean meal (48% CP) Lard Dicalcium phosphate Limestone Salt Vitamin premix b Selenium premix c L-Lysine HCl Tylan d CuSo 4 5H 2 O e FeSO 4 7H 2 O f MnSO 4 H 2 O g KI h ZnSO 4 H 2 O Phytase i Cornstarch Calculated nutrient composition DE, kcal/kg 3,485 3,485 3,485 3,485 CP, % Lysine, % Ca, % P, % Mg, g/kg Cu, mg/kg Mn, mg/kg Zn, mg/kg a Phytase units per kilogram of diet, calculated as the amount of enzyme that liberates 1 mmol of inorganic phosphorus per minute from sodium phytate at ph 5.5 and 37 C. Provided per kilogram of diet: vitamin A, 6,108 IU; vitamin D 3, 600 IU; vitamin E, 23 IU; menadione sodium bisulfite, 1.2 mg; vitamin B 12,31mg; riboflavin, 6 mg; d-pantothenic acid, 22.5 mg; niacin, 35 mg. Provided per kilogram of diet: Se, 300 mg. Provided 10.4 mg tylosin phosphate per kilogram of diet. Provided per kilogram of diet: Cu, 10 mg; Fe, 100 mg; Mn, 27.5 mg; I, 1.4 mg. Contains 5,000 units of phytase activity per gram of premix and supplies 1,500 phytase units per kilogram of diet. Materials and Methods Dietary Treatments. Four 18% CP diets (Table 1) were formulated to contain either 0 or 100 mg of supplemental Zn per kilogram with or without the addition of 1,500 phytase units ( PU) per kilogram. The phytase (Natuphos ) was produced by a recombinant Aspergillus niger. One phytase unit is defined as the amount of enzyme necessary to liberate 1 mmol of inorganic phosphorus per minute from sodium phytate at ph 5.5 and 37 C. The diets that were not supplemented with phytase contained less than 60 PU/kg, and the phytase-supplemented diets contained 1,510 and 1,560 PU/kg by analysis. Diets were calculated to contain.87% phytic acid. All nutrients, except Zn, were supplied in the diet to meet or exceed current recommendations (NRC, 1988) for the 10-kg pig. The diets were not pelleted. Growth Performance. Pigs were weaned at approximately 26 d of age and fed a 22% CP, 1.15% lysine diet for 5 d, at which time pigs for the experiment were selected. Forty-eight pigs (barrows:gilts, 1:1) with an average initial weight of 9.4 kg were used to determine the influence of dietary phytase and supplemental Zn on the utilization of minerals; growth performance and plasma mineral concentration served as the response criteria. Pigs were individually housed in.86-m.38-m pens on elevated, plastic-coated expanded metal floors in an environmentally controlled room where temperature was maintained between 24 and 26 C. The existing plumbing was modified to circulate deionized water from a reservoir through polyvinyl chloride pipes. Samples of deionized water were analyzed and found to contain.08 mg of Zn/L. Pigs were blocked by sex and initial weight and assigned to dietary treatment. Feed was withheld from the pigs for approximately 24 h before the start of experiment but water was available for ad libitum consumption. At the initiation of the experiment, pigs were weighed, and 7 ml of blood was collected from the anterior vena cava into heparinized tubes and centrifuged at 2,500 g for 20 min to obtain plasma

3 3386 ADEOLA ET AL. Table 2. Analyzed mineral composition of corn, soybean meal, and diets used in the study a (as-fed basis) Soybean Supplemental Zn, mg/kg: Item Corn meal Phytase, PU/kg: 0 1, ,500 Ca, % P, % Mg, % Cu, mg/kg Mn, mg/kg Zn, mg/kg a The analyzed ( % ) crude protein and indispensable amino acids of corn and soybean meal, respectively were: CP, 7.1 and 47.9; Arg,.32 and 3.45; His,.23 and 1.22; Ile,.26 and 2.11; Leu,.85 and 3.62; Lys,.24 and 2.86; Met,.18 and.68; Phe,.32 and 2.32; Thr,.25 and 1.79; and Val,.30 and Diets for subsequent mineral analysis. Pigs were allowed ad libitum access to feed for 21 d; pig weight and feed intake were recorded weekly. On d 21, after a 24-h fast, pigs were weighed and bled as described above. Plasma samples were stored at 18 C until they were analyzed for mineral concentrations. Barrows were immediately transferred to metabolism cages for use in the following mineral balance study. Animal care and experimental protocols were approved by the Purdue University Animal Care and Use Committee. Mineral Balance. The 24 barrows from the growth performance study were used in a mineral balance study consisting of a 4-d diet adjustment period and a 4-d total fecal and urine collection period. Pigs had free access to deionized water and were fed 500 g of diet (Table 1) per feeding at 0800 and 1500 during the adjustment and collection periods. In the mineral balance study, pigs continued on the same diets they received prior to being moved into metabolism crates. Feces were collected following each feeding, weighed, composited, and stored at 18 C. Urine volume was measured and recorded daily and a 30% aliquot was taken. Ten milliliters of formaldehyde was added to the urine collection vessels daily. All samples were stored at 18 C until immediately before processing. Feces from each pig were thoroughly mixed, and duplicate samples were dried at 55 C and ground in a Wiley mill through a 1-mm screen before mineral analysis. Urine samples were composited within pen and strained through glass wool before mineral analysis. Mineral Analysis. Samples of corn, soybean meal, feed, feces, urine, and plasma were analyzed for P, Ca, Cu, Mg, Mn, and Zn concentration (Table 2) by Inductively-Coupled Plasma Emission Spectroscopy (ICP) using the following procedure. A known amount of sample was placed in calibrated Folin-Wu digestion tubes and 70% nitric acid was added for predigestion. Tubes were heated in a digestion block until nitric oxide was produced and then allowed to cool for 30 min. Hydrogen peroxide (approximately 3.5 ml, 30% solution) was added and the tube returned to the digestion block until oxygen began to evolve, at which time the tubes were again removed from the digestion block until oxygen evolution subsided. Tubes were then returned to the digestion block for 1 h at 180 C until the volume was reduced to approximately 1.5 ml. When cool, 5% nitric acid was added to bring the tubes to volume. A blank tube was prepared in the same manner. The ICP analysis was conducted using a Perkin Elmer Plasma 400 ICP/AES spectrometer (Norwalk, CT) with an AS 90 autosampler. Sample solutions were placed in 50-mL centrifuge tubes in the autosampler rack with a standard after every 10th sample. Solutions were pumped into a cross flow nebulizer by a peristaltic pump and the resulting aerosol was led into the argon stream going to the plasma torch. A background correction was obtained by measuring the intensity near the desired line, and subtracted from the intensity measured at the peak of the emission line. Standardization of the instrument was conducted at the beginning of each set of determinations. The entire calibration was repeated if one of the standards produced a result that deviated more than 5% from the known value. Statistical Analysis. Data were analyzed as a 2 2 factorial arrangement of treatments (Zn at 0 or 100 mg/kg of diet and phytase at 0 or 1,500 PU/kg of diet) for main effects and interaction of Zn and phytase using the GLM procedure of SAS (1988). Initial plasma mineral concentrations were used as covariates in plasma mineral analysis. The individual pig served as the experimental unit in the study. Results Growth Performance. The addition of 1,500 PU to the diet, regardless of the level of Zn, resulted in an increase ( P <.05) in the rate of body weight gain; the pigs fed diets containing phytase averaged 2.3 kg heavier ( P <.05) at the end of the 21-d experiment (Table 3). A numerical increase of 76 g/d in the rate of body weight gain was observed by supplementing the

4 PHYTASE- AND ZINC-SUPPLEMENTED DIETS FOR PIGS 3387 Table 3. Growth performance of 10-kg pigs fed supplemental zinc with or without phytase a Item and phytase, PU/kg Supplemental dietary zinc, mg/kg x SEM Initial weight, kg , x Final weight, kg b , x Gain, g/d b , x Feed intake, g/d (as-fed) , , x Gain:feed, g:kg b , x a b Data are means of 12 pigs per treatment. Phytase effect significant at P <.05. Table 4. Final (d 21) plasma calcium, phosphorus, magnesium, zinc, iron, and copper concentrations of pigs fed supplemental zinc with or without phytase a Item and phytase, PU/kg Supplemental dietary zinc, mg/kg x SEM Calcium, mg/l , x Phosphorus, mg/l bc , x Magnesium, mg/l d , x Zinc, mg/l bce , x Copper, mg/l , x a Data are means of 12 pigs per treatment. Initial average plasma concentrations were: Ca, 97 mg/l; P, 117 mg/l; Mg, 18 mg/l; Zn,.94 mg/l; and Cu, 1.52 mg/l. Phytase effect at P <.01. Zinc effect at P <.01. Phytase effect at P <.05. Phytase zinc interaction effect at P <.01. diet containing no phytase with 100 mg of Zn/kg of diet. However, this numerical increase in rate of gain was still substantially lower than the increase in rate of body weight gain obtained when the 0 mg of supplemental Zn/kg diet was supplemented with 1,500 PU (76 vs 155 g/d). Feed intakes were similar ( P >.05) among dietary treatments. Phytase supplementation improved feed efficiency. Plasma Minerals. Initial plasma mineral concentrations were similar across treatment groups and averaged 97, 117, 18,.94, and 1.52 mg/l for Ca, P, Mg, Zn, and Cu, respectively. Plasma Ca concentration was not affected ( P >.05) by dietary treatments (Table 4). Plasma P concentration increased in response to phytase and Zn ( P <.01) independently. The addition of 100 mg of Zn/kg of diet to the diet that was not supplemented with phytase resulted in plasma P concentrations similar to those of pigs that received phytase-supplemented diets with 0 mg of supplemental Zn/kg of diet. Plasma Mg concentrations were unaffected by Zn supplementation and increased ( P <.05) in response to the addition of phytase to the diet. Initial plasma Zn concentrations (.94 mg/l) were similar across treatment. However, plasma Zn concentrations of the pigs fed diets that were not supplemented with phytase or Zn (.35 mg/l) decreased dramatically during the 21-d growth period, possibly indicating that these pigs were marginally Zn-deficient (Table 4). The plasma Zn concentration of the pigs fed the diets with Zn or diets with phytase plus Zn was numerically higher than the plasma Zn concentration at the beginning of the experiment. The addition of Zn to the diet, with or without phytase, resulted in an increase ( P <.01) in circulating Zn levels; the plasma Zn concentration was similar (phytase Zn, P <.01) at 100 mg of supplemental Zn/ kg of diet across phytase treatments. Plasma Cu concentration at the end of the 21-d experiment was numerically higher when supplemental phytase was present in the diet than when diets were not supplemented with phytase. Furthermore, plasma Cu concentration was lower at the end of the 21-d study (1.36 mg/l) than at the beginning of the study (1.52 mg/l), regardless of Zn supplementation. Manganese concentration in the serum was not detectable under the assay conditions (limit of detection was.14 mg/l). Mineral Balance. The results of the mineral balance study indicated that daily Zn retention (Table 5) increased when phytase was added to the diet ( P <.05), regardless of dietary Zn level. Pigs fed diets containing 100 mg of supplemental Zn/kg retained more Zn ( P <.05) than pigs that received no

5 3388 ADEOLA Table 5. Zinc balance of pigs fed supplemental zinc with or without phytase a Item and phytase, PU/kg Supplemental dietary zinc, mg/kg x SEM Zinc, mg/d Intake , x Absorbed b , x Retained b , x a Data are means of six pigs per treatment. b Phytase effect at P <.05; zinc effect at P <.05; phytase zinc interaction effect at P <.1. supplemental Zn; the response was greater with the addition of dietary phytase (phytase Zn, P <.10). The addition of phytase to the diet increased Zn retention ( P <.05) regardless of Zn level. Zinc retention increased from 29.3% of intake for pigs fed diets without phytase to 43 or 48.2% of intake for pigs fed diets containing phytase and supplemental Zn at either 0 or 100 mg/kg, respectively. Intakes of Ca, P, Mg, Mn, and Cu were 7.5, 5.23, and 1.26 g/d and 44.3 and 13.4 mg/d, respectively. Absorption and retention of Ca, P, and Cu increased ( P <.05) when phytase was present in the diet (Table 6). However, addition of phytase did not affect ( P >.05) absorption or retention of Mg and Mn. The main effect of supplemental Zn on Ca absorption and retention was significant at P <.05. Zinc supplementation reduced Mg retention ( P <.05) and severely decreased Cu absorption and retention ( P <.05). The absorption and retention of Mn were reduced ( P <.05) by the addition of 100 mg of supplemental Zn/kg when the diet was not supplemented with phytase, but not when the diet was supplemented with phytase, resulting in a phytase Zn interaction ( P <.05). Discussion The phytic acid present in most grains makes approximately 60 to 80% of the P unavailable for nonruminants due to insufficient phytase enzyme activity in the gastrointestinal tract (O Dell and Savage, 1960; Oberleas et al., 1962). Soybean meal has been reported to have a phytic acid content of between.85 and 1.5% (Nwokolo and Bragg, 1977; Jaffe, 1981). Nwokolo and Bragg (1977) reported that ET AL. Table 6. Calcium, phosphorus, magnesium, manganese, and copper balance of pigs fed supplemental zinc with or without phytase a Item and phytase, PU/kg Supplemental dietary zinc, mg/kg x SEM Calcium, g/d Absorbed bcd , x Retained be , x Phosphorus, g/d Absorbed b , x Retained b , x Magnesium, mg/d Absorbed e , x Retained cd , x Manganese, mg/d Absorbed ce , x Retained ce , x Copper, mg/d Absorbed bce , x Retained bce , x a Data are means of six pigs per treatment. Average mineral intakes were: Ca, 7.49 g/d; P, 5.23 g/d; Mg, 1,260 mg./d; Mn, 44 mg/d; and Cu, 13 mg/d. Phytase effect at P <.05. Zinc effect at P <.05. Phytase zinc interaction effect at P <.10. Phytase zinc interaction effect at P <.05. the phytic acid content of soybean meal was, however, lower than other protein sources for nonruminants. Furthermore, O Dell and Savage (1960) initially reported that the phytic acid in soybean meal not only rendered a large portion of the total P unavailable, but

6 PHYTASE- AND ZINC-SUPPLEMENTED DIETS FOR PIGS 3389 also, due to its high affinity for Zn, could effectively bind sufficient Zn to decrease growth rates in chicks. Similar effects of phytate on Zn deposition and growth rate have been reported by Likuski and Forbes (1964) for chicks and by Davies and Nightingale (1975) for rats. Davies and Olpin (1979) suggested that a Zn- Ca-phytate complex formed in the upper gastrointestinal tract renders greater than 90% of dietary Zn unavailable. Davies and Nightingale (1975) also indicated that in the absence of phytate approximately one-third of the endogenously secreted Zn is normally reabsorbed; however, with inclusion of phytate in the diet, approximately 80% of the endogenously secreted Zn is rendered unavailable. Thus, by reducing the availability of dietary and endogenously secreted Zn, phytate may increase the rate at which endogenous Zn is lost from the body, thereby contributing to a reduction in growth rate. Results of the present experiment indicate that the hydrolysis of phytate by the addition of 1,500 PU/kg to a corn-soybean meal diet can result in an increase in the availability of sufficient nutrients to effect an increase in the rate of body weight gain. The numerical response in ADG to increasing supplemental Zn in the diets without phytase indicated that Zn may be partially responsible for the increased growth rate. Furthermore, the increased Zn retention, as well as the higher plasma Zn concentration, observed with dietary phytase supplementation in this experiment seemed to be due to the ability of phytase to liberate sufficient Zn from Zn-phytic acid complexes, thereby increasing zinc availability and maintaining normal circulating Zn concentrations. The increased growth response (Table 3) to phytase supplementation in the present studies could be due to the overall increase in plasma P, Mg, and Zn (Table 4); increase in Zn absorption and retention (Table 5); and increase in Ca, P, and Cu absorption and retention (Table 6). Urinary Ca excretion (difference between absorbed and retained) was.33 g/d in pigs that did not receive phytase supplementation, and urinary P excretion was negligible (.01 g/d). Hypercalcuria and hypophosphaturia are signs of P deficiency and might be related to insufficient availability of P. Pallauf et al. (1994a,b) reported similar findings in pigs fed corn-soybean meal-based or wheatbarley-peas-field beans-based diets. Thus, the improved growth rate in pigs fed diets supplemented with phytase in the present experiments could also be due to an amelioration of P deficiency. The results of the present experiment also support the recent study of Han et al. (1994) that indicated that the degree of solubility of Zn in vitro was dependent not only on the presence of phytate, but was inversely related to the degree of phosphorylation of the phytate molecule, ranging from 93% soluble in the presence of inositol triphosphate ( IP 3 ) to only 8% soluble in the presence of inositol hexophosphate ( IP 6 ). Xu et al. (1992), using methods different from those of Han et al. (1994), observed similar, but more dramatic, effects of phytate phosphorylation state on Zn solubility; less than 20% of the Zn was soluble in the presence of IP 3. The addition of phytase to cornsoybean meal diets has been shown by Jongbloed et al. (1992) to decrease the duodenal phytic acid content and ileal levels of inositol tetraphosphate ( IP 4 ), inositol pentaphosphate ( IP 5 ), and IP 6, indicating that 60 to 74% of the phytic acid was degraded by the terminal ileum when phytase was added to the diet, compared with only 10% degradation when phytase was not added to the diet. The efficacy of phytase in improving P digestibility was recently shown to be dependent on dietary Ca concentration (Lantzsch et al., 1995). Previous experiments (Jongbloed et al., 1992; Xu et al., 1992; Han et al., 1994; Pallauf et al., 1994a), along with the increases in growth rate, plasma P and Zn concentrations, and P and Zn retention observed in the present study, suggest that phytase is effective in dephosphorylating the phytate molecule, thus increasing the availability and utilization of nutrients for growth. Furthermore, increases in apparent absorption and retention of Ca indicate that Ca in the Zn-Caphytate complex (Davies and Olpin, 1979; Xu et al., 1992) is liberated upon phytase addition (Nasi, 1990; Young et al., 1993). Previous research, however, has attributed the improvements in growth rate with phytase addition to increased P availability and utilization alone (Nelson et al., 1971; Simons et al., 1990; Cromwell et al., 1993; Lei et al., 1993b,c). In studies with pigs fed corn-soybean meal diets from 9 to 25 kg live weight, Pallauf et al. (1992) observed increased Zn absorption in phytase-supplemented diets. Results of the present study show that growth rate improvements arising from dietary phytase supplementation may be due to an overall increase in mineral availability, particularly for Ca, P, and Zn. Davies and Nightingale (1975) reported that the presence of phytate in the diet had detrimental effects on Cu retention. This is potentiated by phytate reportedly having the highest (Vohra et al., 1965), or second-highest (Maddaih et al., 1964) affinity for Cu and phytate having one more binding site for Cu than for Zn (Champagne and Hinojosa, 1987). Phytase supplementation of diets was reported to improve Cu absorption in weanling pigs (Pallauf et al., 1992). The improvement in Cu absorption and retention as well as the slight increase in plasma Cu concentration observed in the present experiment further suggest that phytase is effective in liberating minerals from the mineral-phytate complex, thus increasing the availability and utilization of minerals for growth. Brink et al. (1991) observed that phytate in soybean meal reduced Mg digestibility. This reduction in Mg digestibility was later shown to occur through an increase in endogenous Mg losses (Brink et al.,

7 3390 ADEOLA 1992). Although plasma Mg concentrations in the present experiment indicate phytase addition may have improved Mg availability, differences in apparent absorption and retention were not detected. The increases in plasma Mg concentrations indicate that although digestibility of Mg may be unaffected by phytase addition, circulating concentrations of available Mg may be increased due to reduced endogenous losses of Mg from the intestine as a result of decreased phytate concentrations (Brink et al., 1992). Pallauf et al. (1992) reported that phytase supplementation of diets improved Mg absorption, but in more recent experiments, phytase-induced improvement in Mg absorption could not be demonstrated (Pallauf et al., 1994a,b). Davies and Nightingale (1975) reported that phytate reduced retention of Mn; however, the binding affinity of phytate for this mineral is low compared with the affinity for other minerals (Maddaih et al., 1964; Vohra et al., 1965; Oberleas, 1973). The data from this experiment indicate that phytase was not effective in increasing Mn availability. Initial plasma Zn concentrations (.94 mg/l) were within normal limits (Ullrey, 1967). However, by the conclusion of the experiment, the pigs fed the diet without phytase and 0 mg of supplemental Zn/kg had lower plasma Zn concentration (.35 mg/l) than pigs on the other three treatments (.83, 1.21, 1.21 mg/l). This observation indicated that the pigs receiving the diet without supplemental Zn or phytase were in a marginal zinc status. Final plasma Zn concentration indicates that the addition of available Zn, either through Zn supplementation or phytase addition, dramatically increased Zn availability, with supplemental Zn causing circulating Zn concentrations to be slightly above the normal values reported by Ullrey et al. (1967). This confirms the results of Pallauf et al. (1994a,b), who reported that phytase supplementation of diets increased plasma Zn concentration. Supplemental Zn and phytase in combination were unable to further increase circulating Zn concentrations above those obtained by supplementation of Zn alone. Apparent Zn retention without inclusion of phytase in the diet was approximately 29% of Zn intake, regardless of dietary Zn level. However, when phytase was added to the diet, Zn retention increased to 43 and 48% of zinc intake for pigs fed diets containing 0 and 100 mg of supplemental Zn/kg of diet, respectively. These data indicate that phytase is effective in increasing the total availability of Zn and may potentiate Zn uptake when supplemental Zn is present. The failure to observe further increases in circulating Zn concentrations in conjunction with increases in Zn digestibility indicate that the maximum plasma Zn concentrations may not be a good indicator of absolute zinc availability and(or) a reduction in endogenous Zn losses. Plasma P concentrations also responded positively to Zn supplementation, although results of the ET AL. mineral balance study failed to show an effect of Zn supplementation on P balance. Supplemental Zn also had a positive influence on Ca balance, whereas Cu, Mg, and Mn balance were all reduced with Zn supplementation. These data are in agreement with those of Blakeborough and Salter (1987), who observed that at high dietary Zn concentrations the uptake of closely related metals such as Cu may be inhibited. Phytic acid is known to form complexes with protein in the small intestine. A stepwise hydrolysis of phytate-protein complex would be expected to increase nitrogen digestibility (Pallauf et al., 1994b). Supplementation of diets with phytase did not have any effect on apparent fecal digestibility of nitrogen in the present study (data not shown). Similar observations have been reported by Nasi (1990) and Pallauf et al. (1994b). However, fecal nitrogen digestibility data cannot be used to entirely exclude a positive effect of phytase on nitrogen digestibility because of the known influence of hindgut fermentation of amino acids to ammonia and amines in pigs. Implications The results of this experiment indicate phytase is effective in increasing the circulating plasma concentrations of P, Mg, Zn, and Cu. Although plasma Ca concentrations did not increase with addition of phytase to the diet, Ca, P, and Zn absorption and retention were increased by phytase in the mineral balance study. Plasma Cu and Mg concentrations were unaffected by dietary Zn concentration; however, the addition of 100 mg of supplemental Zn/kg of diet resulted in a decrease in the retention of these nutrients, as well as that of Mn. The addition of phytase to the diet seemed to be effective in reducing the inhibitory effects of phytic acid and Zn on Cu and Mn retention. The results of the present studies indicate that phytase is effective in increasing the availability of minerals in the mineral-phytic acid complex for absorption and utilization for growth. Literature Cited Blakeborough, P., and D. Salter The intestinal transport of zinc studied using brush-border-membrane vesicles from the piglet. Br. J. Nutr. 57:44. Brink, E. J., P. R. Dekker, E.C.H. van Beresteijn, and A. C. Beynen Inhibitory effect of dietary soybean protein vs. casein on magnesium absorption in rats. J. Nutr. 121:1374. Brink, E. J., G. J. van der Berg, R. van der Meer, H.T.H. Wolterbeck, P. R. Dekker, and A. C. Beynen Inhibitory effect of soybean protein vs. casein on apparent absorption of magnesium in rats is due to greater excretion of endogenous magnesium. J. Nutr. 122:1910. Champagne, E. T., and O. Hinojosa Independent and mutual interactions of copper(ii) and zinc(ii) ions with phytic acid. J. Inorg. Biochem. 30:15.

8 PHYTASE- AND ZINC-SUPPLEMENTED DIETS FOR PIGS 3391 Cromwell, G. L., T. S. Stahly, R. D. Coffey, H. J. Monegue, and J. H. Randolph Efficacy of phytase in improving the bioavailability of phosphorus in soybean meal and corn-soybean meal diets for pigs. J. Anim. Sci. 71:1831. Davies, N. T., and R. Nightingale The effects of phytate on intestinal absorption and secretion of zinc, and whole-body retention of zinc, copper, iron and manganese in rats. Br. J. Nutr. 34:243. Davies, N. T., and S. E. Olpin Studies on the phytate:zinc molar contents in diets as a determinant of Zn availability to young rats. Br. J. Nutr. 41:590. Han, O., M. L. Failla, A. D. Hill, E. R. Morriss, and J. C. Smith, Jr Inositol phosphates inhibit uptake and transport of iron and zinc by a human intestinal cell line. J. Nutr. 124:580. Jaffe, G Phytic acid in soybeans. J. Am. Oil Chem. Soc. 58: 493. Jongbloed, A. W., Z. Mroz, and P. A. 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