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2604 Advances in Environmental Biology, 5(9): 2604-2610, 2011 ISSN 1995-0756 This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Evaluation of the Effects of Natuphos on Ileal Protein Digestibility, Serum Level of Minerals, Intestinal Histology and Performance of Broiler Chicks. 1 P. Moemeni, 2Javad Pourreza, 3Ebrahim Rowghani Haghighi Fard, 4Mohammad Javad Zamiri 1 Department Department 3 Department 4 Department 2 of of of of Animal Animal Animal Animal Science, Science, Science, Science, Shiraz University, Shiraz, Iran. Islamic Azad University, Shahrekord Branch, Shahrekord, Iran. Shiraz University, Shiraz, Iran. Shiraz University, Shiraz, Iran. P. Moemeni, Javad Pourreza, Ebrahim Rowghani Haghighi fard, Mohammad Javad Zamiri: Evaluation of the effects of natuphos on ileal protein digestibility, serum level of minerals, intestinal histology and performance of broiler chicks. ABSTRACT The goal of the study was to investigate the effect of graded levels of phytase (Natuphos) on ileal digestibility and intestinal histology of broiler chicks. Three hundred seventy five (375) one day old broilers (A Plus strain) were randomly divided into 25 groups, 15 chicks per group. Five diets were formulated and each diet was given to five groups (replicates) for 42 day of age. Two phased diet were used. The starter and grower diets were fed from 0-21 days and 21-42 days of age respectively. Experimental diets (2-5) were formulated to contain 30% available phosphorus less than NRC recommendations. These diets (2 to 5) were supplemented with Natuphos and contained 150-600 FTU/Kg of exogenous phytase respectively. Reduction in dietary available P reduced villi length consequent of that was lower performance. Added phytase improved ileal protein digestibility, blood parameters, villi length, crypt depth and performance. The results of present study suggest improvement in ileal histology, which causes better nutrients absorption and consequently better performance due to supplemental phytase, and the best effect was observed between 450- 600 FTU/Kg diet. Key words: natuphos; ileal protein digestibility; blood parameters; performance; intestinal histology; broiler. Introduction Phytate is a complex compound which contains bounded phosphorus and other minerals or nutrients, and it is found in most plants, such as corn and soybean. Corn and soybean make up an important portion of diet for chickens; much of the phosphorus in these diets is unavailable for absorption [24]. Almost 61-70% phosphorus launch in corn-soybean meal diet is in the form of phytate phosphorus [26]. When we use diet contain corn or other cereals, chicken cannot utilize phytate phosphorus and this phosphorus is excreted and leading to environmental pollution. Natuphos is produced from fungi Aspergillus Nijer. This enzyme is a type of microbial phyatse that release components bounded with phytate. Microbial phytase has taken considerable attention as a feed additive for pigs and poultry [27]. Therefore, the unavailable nutrients will be available and bird can use them. Small intestine is the major site for secretion of digestive enzymes, but bird devoid of phytase secretion. The gastrointestinal tract (GIT) can be reasoned as a microbial ecosystem, the complexity of which most obviously depends on the GIT anatomy, therefore health of GI and its proper functioning influence general performance and health of flock [7]. The goal of this study was to detect the effect of microbial phytase on ileal digestibility and Corresponding Author Parichehr Moemeni, Department of Animal Science, Shiraz University, Shiraz, Iran E-mail: [email protected] Mobile:+98-0912-60-65-770 2605 Adv. Environ. Biol., 5(9): 2604-2610, 2011 intestinal histology of broiler in light microscope and measurement parameters with DINO software. Materials and methods Three hundred seventy five-day-old broiler chickens (A plus strain), with equal number of males and females, were selected and randomly allocated to 25 groups, five groups per dietary treatment with 5 repeated. Two-phased diets were used. The starter and grower diets were fed from 0 to 3 weeks and 4 to 6 weeks of age respectively. Experimental diets (2-5) contained 30% available P less than NRC [13] recommendations (Table 1). Diets 2 to 5 were supplemented with 150, 300, 450 and 600 FTU/Kg phytase from natuphos. Feed and water were provided ad libitum. Ileal protein digestibility, performance, blood parameters (P, Zn and Cu) and ileal histology were measured. On the morning of d 21 and 42, 150 chicks (6 chicken per pen with 5 pens per treatment) were killed by neck dislocation. All of the ileal digesta between Mecel diverticulum and the terminal ileum (2 cm above the ileo-cecal junction) were obtained immediately and carefully, as explained by Huang et al. [8]. The digesta of the 6 birds per pen was pooled as one sample into a plastic bag and immediately stored at -20°C. Digesta were freezedried, grounded through a 1.00-mm mesh screen, and mixed thoroughly before analyses. Ileal protein digestibility was measured by AIA (Acid Insoluble Ash) as marker [21]. At d 42, 75 chicks (3 per pen with 5 pens per treatment) were chosen and sample of their blood were gathered. Blood samples were centrifuged and stored at -20°C. Phosphorus concentration was determined by phosphorus kit [15]. Zinc and copper concentration were measured by Atomic Absorption (AOAC 3]. At the end of d 21 and 42, 150 chicks (6 per pen with 5 pens per treatment) were weighted and average daily gain (ADG), feed intake, and feed conversion were calculated. At the end of 3th and 6th weeks, six chickens from each treatment were neck dislocated and ileal contents were emptied and samples of ileum were taken. The samples were flushed with physiological saline and fixed in 10% formalin. Intestinal sample were then prepared after staining with hematoxylin and eosin using standard paraffin embedding procedures. Villus height was measured from the tip of the villi to the villus crypt junction, and crypt depth was defined as the depth of the invagination between adjacent villi. Villus tip wide, villus bottom wide, distance between villus and epithelium wide was measured by DINO software. Statistical Analyses: Intestinal histology data was analyzed by the General Linear Model (GLM) procedure of SAS software [19]. Differences among means were tested using SNK test. A significance level of 0.05 was used. Results: Ileal Protein digestibility was improved significantly with increasing enzyme level, (P<0.05) both in distal and proximal ileum at 21 and 42 days of ages (Table 2). Table 3 indicates the effect of added Phytase on body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) of chickens at 21 and 42 days of age. There was no significant difference in feed intake at 21 and 42 days of age due to supplemental of phytase. Body weight gain was increased significantly (P<0.05) only at 600 FTU/Kg phytase in day 21. There was no significant difference in body weight gain during 21-42 days of age due to added phytase A significant improvement was observed in feed conversion ratio both in 21 and 42 days of age due to supplemental phtase and it was lowest at 600 FTU/Kg phytase Table 4 shows the effect of supplemental Phytase on body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) during 0 to 42 days of age. Although, BWG of treatment 5 which received 600 FTU/Kg phytase was the highest, but there was no significant difference in BWG between treatments in this period. There were no significant differences in FI. Added supplemental phytase improved FCR significantly (P<0.05) and the best FCR belonged to 600 FTU/Kg phytase. The effect of supplemental phyatse on serum Cu, Zn and phosphorus is presented in table 5. Serum Cu was reduced due to added phytase significantly (P<0.05) as compare to control diet. There was no significant difference in serum Cu, between treatments contained supplemental phytase. Serum Zn was increased significantly (P<0.05) at 450 FTU/Kg phytase. Supplemental phytase at 600 FTU/Kg increased serum phosphorus significantly (P<0.05). The effect of supplemental phyatse on intestinal histology at d 21 is shown in table 6. The chicken fed low phosphrous diet and 150 FTU/Kg phyatse had shorter ileal villus in day 21 compare to other treatments (Figure 1). Crypt depth was significantly (P<0.05) deeper in chickens fed diet with 450 g/ton Natuphos versus control. Ratio of villi height to crypt depth was significantly different (P<0.05) in treatments which received 150 and 450 FTU/Kg diet. Villi wide tip was significantly (P<0.05) wider in control diet in comparison to other treatments Villi wide bottom was significantly (P<0.05) broader in control in comparison to 300 and 450 g/ton natuphos. There was no evidence that natuphos affect distance between villus and epithelium (Figure 1). Adv. Environ. Biol., 5(9): 2604-2610, 2011 2606 Table 1: Composition of experimental diet. Ingredients(%) starter Grower ---------------------------------------------------------------- ------------------------------------------------------------------------1 2 3 4 5 1 2 3 4 5 Corn 59 59 59 59 59 61.8 61.8 61.8 61.8 61.8 Soybean 36.5 36.5 36.5 36.5 36.5 33.5 33.5 33.5 33.5 33.5 Oil 0.8 0.8 0.8 0.8 0.8 1 1 1 1 1 Zeolite 0.23 0.23 0.23 0.23 0.5 0.79 0.79 0.79 0.79 Natuphos 0.015 0.030 0.045 0.060 0.015 0.030 0.045 0.060 1.3 1.62 1.62 1.62 1.62 1.43 1.67 1.67 1.67 1.67 CaCo3 Dicalcium 1.52 0.87 0.87 0.87 0.87 1 0.47 0.47 0.47 0.47 phosphate Mineral 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 supplement Vitamin 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 supplement Salt 0.23 0.23 0.23 0.23 0.23 0.24 0.24 0.24 0.24 0.24 D-L 0.15 0.15 0.15 0.15 0.15 0.3 0.3 0.3 0.3 0.3 Methionine Metabolisabl 2390 2390 2390 2390 2390 2940 2940 2940 2940 2940 e Energy (Kcal/ Kg) Protein (%) 21 21 21 21 21 19.3 19.3 19.3 19.3 19.3 Calcium (%) 0.9 0.9 0.9 0.9 0.9 0.82 0.82 0.82 0.82 0.82 Available 0.41 0.31 0.31 0.31 0.31 0.32 0.23 0.23 0.23 0.23 Phosphorus (%)1 Lysine (%) 1.14 1.14 1.14 1.14 1.14 1.06 1.06 1.06 1.06 1.06 D-L 0.483 0.483 0.483 0.483 0.483 0.348 0.348 0.348 0.348 0.348 Methionine (%) Met+Cys 0.81 0.81 0.81 0.81 0.81 0.68 0.68 0.68 0.68 0.68 (%) 1- Available phosphorus of diets 2 to 5 were 30% less than required phosphorus. Amount of Natuphos added was 150, 300, 450 and 600 gr per ton to diets 2 to 5, and diets had 150, 300, 450 and 600 FUT/Kg phytase. Table 2: Effect of Natuphos on ileal protein digestibility at 21 and 42 days of age Treatment Natuphos 21days 42days (gr/kg feed) -------------------------------------------------------------------------------------------------Proximal Distal Proximal Distal 1(control) 0 71.23Bce1,2 76.86Ade 70.13Ab 73.89Ab 2 150 66.72Ad 70.02Ac 68.22Ab 72.25Ab 3 300 73.96Bae 79.99Abe 71.28Bb 84.12Aa 4 450 75.51Bac 83.57Aab 78.82Ba 85.52Aa 5 600 76.79Ba 85.88Aa 79.03Ba 85.68Aa SEM 1.257 1.257 3.159 3.159 1- There is no significant difference (P>0.05) between means in each column with identical small letters. 2- There is no significant difference (P>0.05) between means in each row with identical small letters Table 3: Effect of Natuphos on body weight gain, feed intake and feed conversion ratio during starter and grower. Treatment Natuphos Starter Grower (gr/kg feed) 21 days of age 42 days of age -------------------------------------------------------------- ------------------------------------------------------------------FI (g) BWG(g/h/d) FCR FI (g) BWG(g/h/d) FCR (feed/gain) (feed/gain) 1(control) 0 0.706a1 0.335a 2.114a 1.882a 1.100a 1.714ab 2 150 0.707a 0.340b 2.079a 1.935a 1.038a 1.871a 3 300 0.680b 0.309b 2.208a 1.860a 1.047a 1.784ab 4 450 0.698a 0.339b 2.066a 1.852a 1.160a 1.601b 5 600 0.702a 0.394b 1.789b 1.860a 1.172a 1.592b SEM 0.00060122 0.00480325 0.12327982 0.0568 0.0191 0.0719 1- There is no significant difference (P>0.05) between means in each column with identical small letters. Table 4: Effect of Natuphos on body weight gain, feed intake and feed conversion ratio during starter and grower period. Treatment Natuphos (gr/kg feed) FI (g) BWG(g/h/d) FCR (feed/gain) 1(control) 0 1 2.540 b 1.440 a 1.770 abc 150 2 2.706 a 1.386 a 1.957a 300 3 2.621 ab 1.420 a 1.846ab 450 4 2.405 b 1.464 a 1.649c 600 5 2.400 b 1.493 a 1.617c SEM 0.0926 0.0077 0.085 1- There is no significant difference (P>0.05) between means in each column with identical small letters. 2607 Adv. Environ. Biol., 5(9): 2604-2610, 2011 Table 5: Effect of Natuphos on blood parameters (P, Zn and Cu) of broiler at 42 days of age. Treatment Natuphos (gr/kg feed) P(mg percent) Zn(μ/ml) Cu(μ/ml) 1(control) 0 0.310b(1) 3.706b 0.560a 2 150 0.296b 3.541b 0.159b 3 300 0.307b 3.791ab 0.147b 4 450 0.307b 4.820a 0.165b 5 600 0.339a 4.543ab 0.146b SEM 0.0012 1.641 0.0811 There is not significant difference between means which have similar letter or letters within a column (SNK test, P>0.05). A=villi height (mm) B=Crypt depth (mm) E= Distance between villus (mm) C= Villi wide tip (mm) D=villi wide bottom (mm) E= Distance between villus (mm) A=villi height (mm) B=Crypt depth (mm) C= Villi wide tip (mm) D=villi wide bottom (mm) Fig. 1: Comparison of villus at 21 day of age and Measuring of parameters by DINO software (200x). Adv. Environ. Biol., 5(9): 2604-2610, 2011 2608 Table 6: Effect of Natuphos on intestinal histology (Ileum) of broiler at day 21 Treatment Natuphos villi Crypt Villi wide villi wide Distance Epithelium Ratio of villi (Kg per height depth tip bottom between (m m) height to (mm) (mm) (mm) (mm) villus(mm) crypt depth ton)1 1(control) 0 1.046a(1) 0.292c 0.230a 0.308a 0.038a 0.041a 3.624a 2 150 0.673b 0.525b 0.125b 0.220ab 0.041a 0.046a 1.300c 3 300 1.071a 0.434bc 0.100b 0.171b 0.039 a 0.050a 2.470b 4 450 1.046a 0.723a 0.096b 0.139b 0.053a 0.043a 1.523c 5 600 1.037a 0.427bc 0.122b 0.256ab 0.038a 0.041a 2.508b SEM 0.140 0.103 0.004 0.009 0.0001 0.000044 2.573 1) There is not significant difference between means which have similar letter or letters within a column (SNK test, P>0.05). Fig. 2: Comparison of villus at 42 day of age. Table 7 indicates the effect of phytase on intestinal histology at day 42. Villous height was significantly (P<0.05) affected by added phytase. Supplemental phytase increased villous height at levels of 300 to 600 FTU/Kg (Figures 2). Other histology parameters were improved due to supplemental phytase. Discussion As expected decreasing available phosphorus in the diet caused a reduction in the performance of chicken. The results obtained in this experiment are in agreement with finding of Punna and Roland [18] and Afsharmanesh and Pourreza [1]. Possitive effect of phytase on performance in broiler has been reported by other investigators (Manangi and Coon [10]; Namkung and Leeson [12]; Pourreza and Classen [17]; Juanpere et al., [9] and Guggenbuhl and Simoesnunes et al., [5]. One of adverse effects of phytate is redution in digestive enzymes activity. Increased ileal protein digestibility was observed with increasing supplemental phytase, which indicated can increase in phytate hydrolysis and consequently reduced it's adverse effect on digestive enzymes, which result better protein digestibility (Pourreza and Classen [17]; Hassanabadi et al., [6]; Selle et al., [20]. Better FCR without increasing feed intake due to added phytase justifies improvement in ileal protein digestibility. Such improvement in protein digestibility due to supplemental phytase have been reported by Namkung and Leeson [12]; Pourreza and Classen [17]; Afsharmanesh and Pourreza [1]; 2609 Adv. Environ. Biol., 5(9): 2604-2610, 2011 Juanpere et al., [9] and Guggenbuhl and Simoesnunes et al., [5] which are in agreement with the results obtained in this experiment. Higher serum Zn and P, due to supplemental phytase indicated higher phytate hydrolysis and more available Zn and P for the chickens. The results of this experiment support the finding of Mohanna and Nys, [11]; Peter et al., [16]; Viveros et al., [22] and Brenes et al., [4], who reported release of Zn due to supplemental phytase in broiler chickens. The results indicated that Zn is more susceptible to phytic acid, more available to react phytate complex than other mineral. Therefore it is expectable to be released by phytase and more available to the chickens. Release of phosphorus from phytate together with more protein and energy available to cells, helps better growth of intestinal cells, indication of that is increased villi height and crypt depth in the chickens fed on phytase supplemented diets. Crypt is considered as the villus factory and a large crypt point enhances tissue turnover and a high demand for new tissue [25]. More available energy and protein facilitate villus growth and higher nutrient absorption. Therefore phytase, by releasing more nutrients (mineral, amino acids and energy) promote villus growth which has been indicated in the present study (Wang and et al., [23]; Onderci and et al., [14]; AlMarzooqi1 et al., [2]. Overall, the results indicated the supplemental phytase, increases ileal digestibility, and release more Zn, also, 450-600 FTU/Kg diet is sufficient for broiler chickens in order to release and provide most of the phosphorus from phytate. Promotes intestinal cells growth, higher surface area for absorption, better health of GI and consequently better chick performance are the benefit of supplemental phytase. 3. 4. 5. 6. 7. 8. 9. 10. Acknowledgment The authors are grateful to Professor Soghra Gholami (faculty of veterinary school of Shiraz University), and Dr. Mohammadreza Rezvani (faculty of department of animal science of Shiraz University) for their skilled technical assistance. Special thanks to Dr. Amir Vosoughi, Dr. Javad Fazlinia for arrangements and dear all who helped us. 11. 12. References 1. 2. Afsharmanesh, M. and J. Pourreza, 2005. Effects of calcium, citric acid, ascorbic acid, vitamin D3 on the efficacy of microbial phytase in broiler starters fed wheat-based diets I. performance, bone mineralization and ileal digestibility. Inter. J. Poult. Sci., 4: 418-424. Al-Marzooqi1, W. and S. Leeson, 2000. Effect of dietary lipase enzyme on gut morphology, gastric motility, and longterm performance of broiler chicks. Poult. Sci., 79: 956–960. 13. 14. AOAC., 2003. Official Methods of Analysis. 17th ed. Association of Official Analytical Chemists, Gaithersburg, MD.USA, pp: 20-33. Brenes, A., A. Viveros, I. Arija, C. Centeno, M. Pizarro and C. Bravo, 2003. The effect of citric acid and microbial phytase on mineral utilization in broiler chicks. Anim. Feed Sci. Technol., 110: 201-219. Guggenbuhl, P. and C. Simoes Nunes, 2007. Effects of two phytases on the ileal apparent digestibility of minerals and amino acids in ileo-rectal anastomosed pigs fed on a maize–rapeseed meal diet. Livest. Sci., 109: 261–263. Hassanabadi, A., H. Nasiri Moghadam, H. Kermanshahi and M. Danesh Mesgaran, 2009. The effect of microbial phytase on apparent digestibility of crud protein, amino acids, minerals and performance of female broiler chickens. Iranian J. of Animal Sci. Res., 1(2): 9-17. (In persion). Heikkinen, J., J. Vielma, O. Kemilainen, M. Tiirola, P. Eskelinen, T. Kiuru, D. Navia-Paldanius, A. Wright, 2006. Effects of soybean meal based diet on growth performance, gut histology and intestinal microbial of juvenile rainbow trout (Oncorhynchua mykiss). Aquaculture, 261: 259-268. Huang, Y.L., L. Lu, X.G. Luo and B. Liu, 2007. An optimal dietary zinc level of broiler chicks fed a corn-soybean meal diet. Poult. Sci., 86: 2582-2589. Juanpere, J., A.M. Perez-Vendrell and J. Brufau, 2004. Effect of microbial phytase on broilers fed barley-based diets in the presence or not of endogenous phytase. Anim. Feed. Sci. Techol., 115: 265-279. Manangi, M.K. and C.N. Coon, 2008. Phytate phosphorus hydrolysis in broilers in response to dietary phytase, calcium, and phosphorus concentrations. Poult. Sci., 87: 1577-1586. Mohanna, C. and Y. Nys, 1999. Changes in zinc and manganese availability in broiler chicks induced by vegetal and microbial phytases. Anim. Feed Sci. Technol., 77: 241-253. Namkung, H. and S. Leeson, 1999. Effect of phytase enzyme on dietary nitrogencorrected apparent metabolizable energy and the ileal digestibility of nitrogen and amino acid in broiler chicks. Poult. Sci., 78: 1317-1379. Natinal Research Council, 1994. Nutrient Requirements for Poultry. 9th rev. Ed. National Academy prees, Washington, DC. Onderci, M., N. Sahin, K. Sahin, G. Cikim, A. Aydý´n, I. Ozercan, and S. Aydin, 2006. Efficacy of supplementation of -amylase-producing bacterial culture on the performance, nutrient use, and gut morphology of broiler chickens fed a corn-based diet. Poult. Sci., 85: 505–510. Adv. Environ. Biol., 5(9): 2604-2610, 2011 15. Orban, J.I., O. Adeola and R. Stroshine, 1999. Microbial phytase in finisher diets of white pekin ducks: Effect on growth performance, plasma phosphorus concentration, and leg bone characteristics. Poult. Sci., 78: 366–377. 16. Peter, C.M., T.M. Parr, E.N. Parr, D.M. Webel, and D.H. Baker, 2001. The effects of phytase on growth performance characteristic, and bone mineralization of late- finishing pigs fed maizesoybean meal diets containing no supplemental phosphorus, zinc, copper and manganese. Anim. Feed Sci. Technol., 94: 199-205. 17. Pourreza, J. and H.L. Classon, 2001. Effect of supplemental phytase and xylanase on phytate phosphorus degradation, ileal protein and energy digestibility of a corn-soy bean-wheat bran diet in broiler chichens. J. Agric. Sci. Technol., 3: 19-25. 18. Punna, S. and D.A. Roland, 1999. Variation in phytate phosphorus utilization with the same broiler starin. J. Appl. Poult. Res., 8: 10-15. 19. SAS, 2004. SAS User’s Guide: Version 9.1. SAS Institute Inc., Cary, NC. 20. Selle, P. H., V. Ravindran, and G.G. Partridge. 2009. Beneficial effects of xylanase and/or phytase inclusions on ileal amino acid digestibility, energy utilisation, mineral retention and growth performance in wheat-based broiler diets. Anim. Feed Sci. Technol., 153: 303-313. 21. Van Keulen, J. and B.A. Young, 1977. Evaluation of acid-insoluble ash as a natural marker in ruminant digestibility studies. J. Anim. Sci., 44: 282-287. 2610 22. Viveros, A., A. Brenes, I. Arija and C. Centeno. 2002. Effects of microbial phytase supplementation on mineral utilization and serum enzyme activities in broiler chicks fed different levels of phosphorus. Poult. Sci., 81: 1172–1183. 23. Wang, Z.R., S.Y. Qiao, W.Q. Lu and D.F. Li. 2005. Effects of enzyme supplementation on performance, nutrient digestibility, gastrointestinal morphology, and volatile fatty acid profiles in the hindgut of broilers fed wheat-based diets. Poult. Sci., 84: 875-881. 24. Watson, B.C., J.O. Matthews, L.L. Southern and J.L. Shelton, 2006. The effects of phytase on growth performance and intestinal transit time of broilers fed nutritionally adequate diets and diets deficient in calcium and phosphorus. Poult. Sci., 85: 493–497. 25. Xu, Z.R., C.H. Hu, M.S. Xia, X.A. Zhan and M.Q. Wang, 2003. Effect of dietary fructooligosaccharide on digestive enzyme activities, intestinal microflora and morphology of male broilers. Poult. Sci., 82: 1030-1036. 26. Yu, B., Y.C. Jan, T.K. Chung, T.T. Lee and P.W.S. Chiou, 2004. Exogenous phytase activity in the gastrointestinal tract of broiler chickens. Anim. Feed Sci. Technol., 117: 295-303. 27. Zacharias, B., H. Ott and W. Drochner, 2003. The influence of dietary microbial phytase and copper on copper status in growing pigs. Anim. Feed Sci. Technol., 106: 139-148.