Effect of an addition of phytase to diets with variable zinc and low phosphorus content on performance, carcass characteristics and bone mineralization of broilers Y. CUFADAR and Y. BAHTIYARCA Departments of Animal Science, Faculty of Agriculture, University of Selçuk, 42031, Kampüs, Konya Turkey Corresponding address: Yusuf CUFADAR, Selçuk Universitesi, Ziraat Fakültesi, Zootekni Bl. 42031 Kampüs, Konya/TURKEY; e-mail: ycufadar@selcuk.edu.tr, Tel.:+90 332 2232814 SUMMARY A 6-week experiment with 960 unsexed one day-old broiler chicks was conducted to evaluate the effect of microbial phytase (Natuphos 500) on the performance, carcass characteristics and toe ash as a measure of bone mineralization when varying levels of zinc (Zn) and low phosphorus content in diet were used. Twelve treatments consisting of three levels of Zn (40, 60 and 160 mg / kg diet) and four levels of phytase (0, 500, 1000 and 1500 U / kg diet) in 3 x 4 factorial arrangement were used with four replicates of twenty birds each. Increase of the dietary zinc content resulted in a non-significant reduction in the body weight gain (BWG), feed consumption (FC), weights of the carcass, neck, thighs, back + breast and a significant decrease (P< 0.05) in weight of wings. There was also a tendency of increasing feed/gain ratio and toe ash percentage. On the other hand, supplemental phytase induced significant increases of FC during the first (0 to 3 weeks), the second (4 to 6 weeks) periods and during all the experimental duration, and significant decreases of feed/gain ratios (for the second period and for all the experimental period). Carcass weights and their components were also increased by phytase supplementation (p < 0.05-0.01). The differences among supplemental phytase with respect to these parameters have not been significant. In the experiment, significant Zn level x phytase level interactions (P< 0.05) have been observed for BWG. BWG have been significantly improved by phytase addition at three dietary Zn levels whatever the age period considered and the effects of this interaction on the BWG were different during the different age periods. Also, the negative impacts of increasing Zn levels on FC feed/ gain ratio and carcass traits (P> 0.05) have been completely corrected by inclusion of phytase to diets. The results indicate that supplementing microbial phytase to broiler diets with various levels of Zn has improved growth performances and carcass traits. KEY-WORDS : Zinc - phytase - broiler - mineralization - growth - carcass traits. Introduction Zinc (Zn) has been known to be an essential nutrient for animals for many years. It is required for skeleton development, growth, skin growth and integrity, appetite, reproduction, wound healing, immune competence and many biochemical processes [13]. Increasing amount of evidence also suggests that Zn plays a critical role in both cell proliferation and cell survival [12]. Practical poultry diets based on cornsoybean meal provide only a marginal level of Zn relative to requirements, because either the presence of factors such as fiber and phytate limiting Zn absorption or concentrations of other divalent cations complicate the utilization of Zn [31]. Major portion of phosphorus in plant-derived ingredient is RÉSUMÉ Effet d un ajout de phytase dans des régimes alimentaires de teneurs en zinc variables et de faible teneur en phosphore sur les caractéristiques des carcasses et sur la minéralisation osseuse des poulets standards. Par Y. CUFADAR et Y. BAHTIYARCA. 960 poussins de poulets standards âgés de 1 jour ont été utilisés pour évaluer les effets d une supplémentation de 6 semaines en phytase microbienne dans des régimes alimentaires de teneurs en zinc variables sur les caractéristiques des carcasses et la minéralisation osseuse (évaluée par la mesure des cendres obtenues à partir des pattes des animaux). Douze régimes alimentaires ont été testés sur des groupes de 80 poussins (repartis en 4 lots de 20) variant selon les teneurs en phytase (0, 500, 1000 et 1500U/kg d aliment) et en zinc (40, 60, 160 mg/g d aliment). L augmentation de la teneur en zinc a entraîné une diminution du poids total, du gain de poids, de la consommation d aliment (FC), du poids de la carcasse, du cou, des cuisses, du thorax et du dos et des ailes (P< 0.05). En revanche, la phytase a induit une augmentation significative de la FC sur toutes les classes d âge et une diminution significative du rapport nourriture / gain de poids sur la durée totale de l expérimentation et plus particulièrement sur les oiseaux de 4 à 6 semaines. Les poids des carcasses et de leurs composants (P< 0.01 et P< 0.05) ont également été augmentés par la supplémentation en phytase. Néanmoins, l intensité des effets observés n a pas été proportionnelle à la dose d enzyme employée. Lorsque l enzyme a été rajoutée à des régimes riches en zinc, le gain de poids a augmenté significativement (P< 0.05) pour les 3 teneurs en zinc testées avec une intensité variable en fonction de la classe d âge considérée. De même, les effets négatifs du zinc sur la FC et sur les paramètres des carcasses ont été complètement corrigés par l ajout de phytase. Ces résultats mettent en évidence l intérêt d une supplementation en phytase dans des régimes alimentaires quel que soit leur teneur en zinc sur les performances zootechniques et sur les caractéristiques des carcasses des poulets. MOTS-CLÉS : Zinc - phytase - poulet - minéralisation - croissance - carcasse. primarily present in the form of phytate which is the mixed salt of phytic acid and which is unavailable for poultry and pigs, because they have limited capability to utilize phytate phosphorus. Phytic acid as a polyanionic molecule forms insoluble complexes with di- and trivalent cations such as calcium, copper, manganese, magnesium, iron, Zn under the ph conditions of the small intestine and, consequently, reduces their availability to animal [24]. VOHRA at al. [32] and DAVIES and OPLIN [5] reported that phytate has the highest binding affinity for copper, zinc, and manganese. In addition to chelating macro- and micro-elements in complete diets, phytic acid may form complexes with proteins and starches [1]. The binding of phytic acid with protein and starch may also reduce the availability of these nutrients from the diet.
356 CUFADAR (Y) AND COLLABORATOR In many experiments, supplemental microbial phytase has been shown to be very effective for improving phytate phosphorus bioavailability. Although, hydrolyzing phytate also liberates Zn, up to now a few studies have been conducted to determine whether or not adding microbial phytase to diets increases Zn bioavailability in chicks [2, 15, 19, 29, 30, 35]. The results of these experiments have indicated that when phytase alone or in combination with 1.25 (OH) 2 D 3 was added to diet, bioavailability and absorption or retention of Zn were increased. Zn levels used in the experimental diets of these studies were lower than the NRC [16] recommendations for normal growth (40 mg Zn/kg) and for maximal tissue deposition (85 mg Zn/kg) as reported by WEDEKIND et al. [33] and MOHANNA and NYS [15]. Furthermore the results based on short-term research, and available phosphorus levels of experimental diets were generally adequate but the potential effects of phytase or zinc on carcass characteristics of broilers were poorly investigated. In addition limited literature reports have focused on effects of phytase supplementation on carcass characteristics of broilers. SCHI- DELER and FERKET [22] reported that phytase supplementation of female broiler diets significantly increased leg quarter weight but did not affect total carcass yield. The objective of the present experiment was to determine the effect of adding microbial phytase to diets with different levels of Zn on performance, carcass characteristics and bone mineralization in broilers fed practical diets containing low phosphorus over the entire production cycle (6 weeks). Materials and Methods ANIMALS, DIETS AND COLLECTION OF DATA A total of 960 mixed sex one-day-old broilers (Ross 308) were used in the study. The chicks were fed with basal starter and finisher diets (Table I) (containing 37.1 and 35.3 mg Zn/kg, native concentration) based on maize-soybean meal from 0-3 and 4-6 weeks of age, respectively. In another experiment conducted by GÖRGÖLÜ et al. [9] prior to this study, the zinc content of the individual feedstuffs, that were generally used in formulation of poultry ration in Turkey, had Table I : Percentage composition of basal diets (as-fed basis)
EFFECT OF AN ADDITION OF PHYTASE TO DIETS WITH VARIABLE ZINC AND LOW PHOSPHORUS CONTENT 357 been chemically determined and these results were used in formulation of our basal diets. The basal diets in which relatively low Zn were accomplished by adding a Zn-free trace mineral mix to the diets were supplemented with 0, 60 and 120 mg Zn/kg as zinc oxide or with 0, 500, 1000 and 1500 U of phytase/kg diet derived from Aspergillus niger (Natuphos 500 G, Kartal Kimya A.S.). Three levels of Zn and four levels of phytase were arranged in factorial manner and the treatments were replicated four times with 20 birds allocated to each replicate (3 Zn x 4 phytase x 4 replicate x 20 birds per pen = total 960 birds). The chicks of approximately same weight were randomly assigned to among 48 floor pens. The basal diets met recommended nutrient requirements [16] except for those of Zn and available phosphorus (AP). To provide an opportunity for measurable response to phytase additions, AP levels of starter and finisher basal diets were formulated below the current recommendations of 0.45 AP [16], because of the response of phytase supplementation was greater at the lower levels of AP [6, 24]. All ingredients with fine particle (limestone, DCP, salt, salynomycine, lysine, methionine, mineral and vitamin premix and supplemental ZnO and phytase) used in the diets were fully mixed to about five kg of ground corn and then this mix was added to other ingredients and all together was mixed in mixer located in the feed processing center. In addition, birds never refused diets whatever the treatment group. Birds were exposed to continuous light and fed diets in a mash form. Feed and water were available for ad-libitum consumption. Body weights and feed consumption were recorded on a pen basis as weekly intervals. Mortality was recorded daily. At the end of the experiment (at six weeks of age), all chicks were slaughtered at a processing plant. Six killed birds from each replicate were randomly selected, and processed, then the warm carcasses were divided according to method of [21] to the proportions of parts. The left middle toes of the selected birds were obtained and dried and then ashed according to [8]. STATISTICAL ANALYSIS The experiment was designed as 3 (Zn level) x 4 (phytase level) factorial within a randomized complete design. A general linear model (GLM) was used for the analysis of variance of the data [28]. Multi-variance analysis was used to determine whether there were any effect due to Zn and phytase levels and the interaction of these factors over time (as the bird aged). The procedure used was the repeated measure orthogonal polynomial analysis found in the proceeding of GLM of the statistical package of Statistica. This analysis enables the experimenter to asses linear, quadratic or cubic trends over time and the interrelationship of factors of interest [36]. If the treatments were found to be significantly different, then Duncan s multiple range tests was used to determine the differences among treatment of least-square means [7]. Results None performance characteristics was significantly affected by the dietary level of Zn as a main factor. When the dietary Zn levels were increased, body weight gain (BWG) and feed intake tended to decrease for the 0 to 3, 4 to 6 and 0 to 6 weeks periods (Figures 1, 2, 3 and Table II). On the contrary, feed/gain ratio tended to increase in broilers for the same periods (Table II). On the other hand, phytase supplementa- Figure 1 : Effect of dietary zinc and microbial phytase levels on body weight gain in 0-3 week old broilers. Figure 2 : Effect of dietary zinc and microbial phytase levels on body weight gain in 4-6 week old broilers. Figure 3 : Effect of dietary zinc and microbial phytase levels on body weight gain in 0-6 week old broilers.
358 CUFADAR (Y) AND COLLABORATOR Table II : Effect of dietary zinc and microbial phytase on feed intake and feed/gain ratio of broilers during the age periods of 0-3, 4-6 and 0-6 weeks. tion as a main factor induced slight increases of BWG (despite these results were not statistically significant) (Figures 1-3). However, feed consumptions for the periods of 0 to 3, 4 to 6 and 0 to 6 weeks of ages have been significantly increased (P< 0.01) and feed/gain ratios for the 4 to 6 and 0 to 6 weeks periods have been significantly decreased (P<0.01, Table II). There was not significant difference among supplemental phytase contents. Zn by phytase dietary content interactions have been observed for BWG (P< 0.05 to 0.01). For all age periods, high dietary Zn supplementation (100 and 160 mg Zn / kg) significantly decreased BWG and phytase additions at the three dietary Zn contents significantly improved BWG for all age periods (figures 1-3). The intensity of the phytase / Zn interactions on the BWG was different according to the experimental periods. The BWG of broilers fed with diets containing 40 mg Zn / kg (no supplemental Zn) and 500, 1000, 1500 U/kg phytase were always greater than that of broilers fed with diets containing 100 and 160 mg Zn / kg and the same contents of phytase. Besides, the differences among treatment groups were statistically significant, especially for the first period (0 to 3 weeks). When dietary phytase supplementation of birds received 40 mg Zn/kg increased, the magnitude of response increased too. The highest BWG was obtained in chicks fed with diet containing 40 mg Zn/ kg and 1500 U/ kg phytase (Figures 1-3). However, any proportional relation between BWG and phytase supplementation was evidenced on birds received 100 and 160 mg Zn/kg. Although increases in dietary phytase lead to increases in feed intake and generally decrease in feed/gain ratio, non-significant Zn / phytase content interaction for feed intake and feed/gain ratio was observed (Table II). Consequently, a significant decrease of performances of broilers due to increasing dietary Zn from 40 to 160 mg/kg has been prevented by phytase supplementation. In the present experiment, percentage of toe ashes was used as a measure of bone mineralisation. Although dietary Zn supplementation alone, or phytase supplementation as mean factor, or Zn / phytase interaction have not significantly influenced percentage of toe ashes (Figure 4), enhancements of this marker were noticed when dietary Zn and/or phytase supplementation increased; toe ash percentage was highest with 160 mg Zn /kg and 1500 U/kg phytase. The effects of the treatments on carcass and parts weights of carcass were presented on Figure 5 and Table III. Although the differences were not statistically significant, increases of Zn contents in the diets induced decreases of the carcass weight (Figure 5), and of the carcass part weights : however, no statistically significant difference was obtained (Table III) except for wing weight (P< 0.05). On the contrary, phytase additions have significantly increased these parameters (P< 0.01) (Table III). Nevertheless, except for carcass weight, no relation was obtained between phytase supplementation and the magnitude of the observed eleva-
EFFECT OF AN ADDITION OF PHYTASE TO DIETS WITH VARIABLE ZINC AND LOW PHOSPHORUS CONTENT 359 Figure 4 : Effect of dietary zinc and microbial phytase levels on toe ash percentages in 6 week old broilers. Figure 5 : Effect of dietary zinc and microbial phytase levels on carcass weight in 6 week old broilers. tions of carcass part weights. On the other hand, with Zn x phytase interaction, all carcass parameters increased with increasing dietary phytase at three dietary Zn levels although this influence was not statistically significantly proportional to phytase dosages. We can conclude that phytase supplementation has prevented the deleterious effects induced by dietary Zn on carcass traits. Discussion In the present experiment, increasing dietary Zn from 40 to 100 and 160 mg/kg resulted in a decrease in the growth performance and carcass parameters; however, the decreases were not statistically significant. However, this was not surprising based on the inconsistencies in the reports of Zn sta- Table III : Effect of dietary zinc and supplemental phytase on weights of carcass parts in 6 week old broilers.
360 CUFADAR (Y) AND COLLABORATOR tus on growth rate, feed consumption and feed conversion ratio. For example THIEL and WEIGAND [29] reported that dietary Zn levels of 27, 45 and 60 mg/kg had no effect on body weight gain. In another two experiments, BIEHLE et al. [2] and YI et al. [35] reported that when the basal diets containing 13 or 20 mg Zn/kg were supplemented with different levels of Zn, growth performance was significantly increased. One explanation for the failure to obtain significant responses to increases of dietary Zn in this experiment could be that Zn levels of basal starter and finisher diets (37.1 and 35.3 mg /kg, respectively) were adequate for the chick s performance. Indeed, Zn requirement of chicks was reported as 40 mg/kg diet by NRC [16] and levels of basal diets were very closed to recommended level. Also, high Zn ingestion has been observed to depress weight gain and feed consumption [10, 20]. The differences among type of chicks, the levels and source of Zn used in the experiments may be other factors accounting for controversial results. There were only a few studies, which have shown the effect of dietary Zn and phytase levels and their interaction on carcass parameters, in compliance with the observation made by COLLINS and MORAN [4]. They reported that supplemental manganese and/or Zn (sulfate form) did not influence the carcass weight, yield, abdominal fat (%) and carcass defects of male broilers from two diverse broiler strains. However, MCNAUGHTON and SCHUGEL [14] reported that the breast meat yield has increased with dietary Zn supplementation in the form of Zn-methionine. In contrast, YILDIZ and YAZGAN [34] observed a significant decrease in weight of breast meat in broilers when basal starter and finisher diets (88 and 93 mg Zn/kg, recpectively) were supplemented with 60 mg Zn/kg from Zn-methionine and 20 mg Zn/kg from lysine. The inconsistency in these reports might be due to the chemical nature of Zn, palatability of diet and genetic differences [20]. In this study, increasing dietary phytase from 0 to 1500 U/kg resulted in non-significant increase in feed consumption and a significant increase in body weight gain and feed consumption and a significant decrease in feed/gain ratio (P< 0.05, Figures 1-3 and Table II). YI et al. [35] and SCHONER et al. [23] have reported similar results. DENBOW [6] has observed improvements in weight gain and feed consumption when dietary phytase has been increased. However, ROBERSON and EDWARDS [19] reported that body weight and gain/feed ratio did not been affected by basal diet (32 mg Zn/kg) supplemented with 750 or 600 U phytase/kg. There was a significant Zn x phytase interaction for body weight gain (Figures 1-3). Adding phytase to diets containing different levels of Zn significantly improved the body weight gain (P< 0.05 to 0.01) increased feed intake and decreased feed/gain ratio (P< 0.01; Table II). Consequently, adding phytase has prevented adverse effects of increasing dietary Zn (40 to 100 or 160 mg/kg). The improvement of body weight gains resulting from the addition of phytase was probably a consequence of increased feed intake. In agreement, using chicks fed by a glucose-soy concentrate diet (13 mg Zn/kg) with graded levels of added Zn, phytase or di- OH-D 3, BIEHLE et al. [2] reported that both phytase and di- OH-D 3 supplementation increased growth rate. In contrast, ROBERSON and EDWARDS [19] did not consistently observe an improvement in body weight and gain/feed ratio in broilers when 600 to 750 U/kg phytase was added to a corn-soybean diet containing 32 mg/kg Zn. This result may be related to higher available phosphorus content in their basal diet. Several criteria have been used for the estimation of mineral availability by broiler chicks, but many researchers prefer determining growth rate and toe ash content, because of their determination is simple. Zn or phytase contents and Zn / phytase interaction did not affect the toe ash percentages (Figure 4). However, there was a tendency of increasing percentage of toe ashes due to the Zn and phytase supplementation and also, at each level of Zn, percentage of toe ashes was increased by phytase supplementation. This conclusion is supported by the studies of other researchers [19, 35]. Results reported here suggest that, when phytase was added to chicken diets containing different levels of Zn, the performance of chicks and Zn utilization have increased. Adding phytase to basal diets with low available phosphorus has increased feed or nutrient intake, decreased feed/gain ratio and significantly increased weights of carcass and parts of carcass, but there were no differences in carcass traits (except for carcass weight) among supplemental phytase levels. These results are in partially agreement with the findings of SCHIDELER and FERKET [22]. This beneficial effects of phytase could possibly be due to the release of minerals such as calcium and phosphorus [3, 17, 25], Zn [3, 15, 17, 25], magnesium, copper and iron [25] from phytate-complexes. Moreover, because starch and proteins are also included in these complexes, phytase action would also induce an increase of starch digestibility, as suggested by [11], and increased protein availability [18, 26, 27]. As a conclusion, the results of this study indicate that microbial phytase is effective for increasing growth performances and carcass traits in broilers fed with diets based on corn-soybean meal and containing different levels of Zn (not deficiency) and low available phosphorus. If broiler diets are supplemented with phytase, the need for Zn supplementation will decrease and Zn level of the trace mineral premix might be, at least, reduced. References 1. ANGEL, R., APPLEGATE, T. J., ELLESTAD, L. A. and DHANDU, A. S.: Phytic acid chemistry: How important is it for phosphorus digestibility in poultry. Multi-State Poultry Meeting, 2002, May 14-16, Indiana, U.S.A. 2. BIEHL, R.R., BAKER, D.H. and DE LUCAH.F.: La-hydroxylated cholecalciferol compounds act additively with microbial phytases. Animal Feed Sci. and Techn., 1995, 77, 241-253. 3. BRENES, A., VIVEROS, A., ARIJA, I., CENTENO, C., PIZARRO, M. and BRAVO, C.: The effect of citric acid and microbial phytase on mineral utilization in broiler chicks. Animal Feed Sci. and Techn., 2003, 110, 201-219. 4. COLLINS, N.E. and MORAN, E.T.: Influence of Supplemental Manganese and Zinc on Live Performance and Carcass Quality of Diverse Broiler Strains. Journal of Appl. Poultry Res., 1999, 8, 228-235.
EFFECT OF AN ADDITION OF PHYTASE TO DIETS WITH VARIABLE ZINC AND LOW PHOSPHORUS CONTENT 361 5. DAVIES, N.T. and OPLIN, S.E.: Studies on the phytate: Zinc molar contents in diets as a determinant of zinc availability to young rats. Br. J. Nutr., 1979, 41, 590-603. 6. DENBOW, D.M., RAVINDRAN, E.T., KORNEGAY, E.T., YI, Z. and HULET, M.: Improving Phosphorus Availability in Soybean Meal for Broilers by Supplemental Phytase. Poultry Sci., 1995, 74, 1831-1842. 7. DÜZGÜNES, O.: Istatistik Metotlari, 578, A.Ü. Zir. Fak. Yayinlari, A.Ü. Basimevi, Ankara, 1975. 8. FRITZ, J.C. and ROBERTS, Y.T.: Use of Toe Ash As a Measure of Calcification in the chick. Journal of The A.O.A.C., 1969, 51 (3), 591-594. 9. GÖRGÖLÜ, M., KUTLU, H.R., BAYKAL, L., ERDAL, I., TOLAY, I. and ÇAKMAK, I.: Determinant of zinc level in feedstuffs used in Çukurova region, First National Zinc Congress, 643-647, 1998. 10. HENRY, P.R., AMMERMAN, C.B. and MILES, R.D.: Effect of dietary zinc on tissue mineral concentration as a measure of zinc bioavailability in chicks. Nutr. Rep. Int., 1987, 35, 15-24. 11. KNUCKLES, B. E. and BETSCHSRT, A. A.: Effect of phytate and other myo-inositol phosphate esters on alpha-amylase digestion of starch. J. Food Sci., 1987, 52, 719-721. 12. MC DONALD, R.S.: The role of zinc in growth and cell proliferation. J. Nutr., 2000, 130, 15005-15085. 13. MCNAUGHTON, J. L.: Utilization mineral chelates, Multi-state Poultry Meeting, 1992, May, 21-23. 14. MCNAUGHTON, J. L. and SCHUGEL, L. M.: Effect of Feeding Complexed and Inorganic Trace Minerals on Broilers Performance and Breast Meat Yield. Poultry Sci., 1991, 70, Suppl. 1. p. 172. 15. MOHANNA, C. and NYS, Y.: Change in Zinc and Manganese Availability in Broiler Chicks Induced by Vegetal and Microbial Phytases. Animal Feed Sci. and Technology, 1999, 77, 241-253. 16. NATIONAL RESEARCH COUNCIL (NRC): Nutrient Requirements of Poultry, 9th Reviewed Ed., National Academy Press, Washington DC., 1994. 17. QIAN, H., KORNEGAY, E.T. and DENBOW, D.M.: Utilization of phytase phosphorus and calcium as influenced by microbial phytae, cholecalciferol and the calcium total phosphorus ratio in broiler diets. Poultry Sci., 1997, 76: 37-46. 18. RAVINDRAN, S., CABAHUG, S., RAVINDRAN, G and BRY- DEN, W.C.: Influence of microbial phytase an apparent ileal amino acid digestibility of feedstuffs for broilers. Poultry Sci., 1999, 78, 699-706. 19. ROBERSON, K. D. and EDWARDS, H. M.: Effects of 1.25 Dihydroxycholecalciferol and Phytase on Zinc Utilization Broiler Chicks. Poultry Sci., 1994, 73, 1312-1326. 20. SANDOVAL, M., HENRY, P.R., AMMERMAN, C.B., MILES, R.D. and LITTELL, R.C.: Relative bioavailability of supplemental inorganic zinc source for chicks. J. Anim. Sci., 1997, 75, 3195-3205. 21. SAYLAM, S. K. and DOGAN, M.: Etlik Piliç Yetistiriciliginde Yerlesim Sikliginin Performansa Etkileri Üzerine Bir Arastirma, Uluslar Arasi Tavukçuluk Konferansi, 24-26 Mayis, ISTANBUL, 1995. 22. SCHEIDELER, S.E. and FERKET, P.R.: Phytase in broiler rations effects on carcass yields and incidence of tibial dyschondroplasia. J. Appl. Poultry Res., 2000, 9, 468-475. 23. SCHONER, V.F.J., HOPPE, P.P. and SCHWARZ, G.: Vergleich der efekte von mikrobieller phytase und anorg nischem phosphat auf die leistungen und die retention von phosphorus, calcium und rohasche bei masthuhnerkuken in der anfangsmast. J. Anim. Physiol. Anim. Nutr., 1991, 66, 248-255. 24. SCHWARZ, G.: Phytase supplementation and waste management. Proceedings of Meeting Arkansas Nutr. Conf., 1994, Sept. 13-15, Fayetteville. pp. 18-51. 25. SEBASTIAN, S., TOURCHBURN, S.P., CHAVEZ, F.R. and LAGUE, P.C. : 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. Poultry Sci., 1996, 75, 729-736. 26. SEBASTIAN, S., TOURCHBURN, S.P., CHAVEZ, F.R. and LAGUE, P.C. : Apparent digestibility of protein and amino acid in broiler chickens fed a corn -soybean diet supplemented with microbial phytase. Poultry Sci., 1997, 76, 1760-1769. 27. SELLE, P. H., RAVINDRAN, V., COLDWELL, R. A. and BRY- DEN, W. C. : Phytate and phytase ; consequences from protein utilazation., Nutr. Res. Rev., 2000, 13, 255-278. 28. STATISTICA.: Statistica For Windows, 5.0 Released, Statsoft Inc. East 13 th Street, Tulsa, USA, 1995. 29. THIEL, U. and WEIGAND, E.: Influence of Dietary Zinc and Microbial Phytase Supplementation on Zn Retention and Zn Excretion in Broiler Chicks. Proceeding of The World s Poultry Congress, 1992, Vol. 3, 460, World s Poultry Sci. Assoc. Amsterdam, the Netherlands, pp. 460. 30. THIEL, U., WEIGAND, E., HOPPE, P.P. and SCHÖNER, F.J.: Zinc retention of broiler chickens as effected by dietary supplementation of zinc and microbial phytase. Eight Int. Symp. on Trace Elements. Trace Elements in Man and Animals, TEMA, 1993. 8, pp. 658-659. 31. UNDERWOOD, E.J. and SUTTLE, N.F.: The mineral nutrition of livestock, 3rd Edition, Moredun Research Institute, Midlothian, UK. pp. 477-512, 1999. 32. VOHRA, P., GRAY, A. and KRATZER, F.H.: Phytric acid-metal complexes, Proc. Soc. Exp. Biol. Med., 1965, 120, 449. 33. WEDEKIND, K. J., HORTIN, A.E. and BAKER, D. H.: Methodolgy for assessing zinc bioavailability: efficacy estimates for zinc-methionine, zinc sulfate and zinc oxide. J. Anim. Sci., 1992, 70, 178-187. 34. YILDIZ, A. Ö. and YAZGAN, O.: Broyler Rasyonlarina Ilave Edilen Çinko-Metiyonin ve Çinko-Lizinin Besi Performansi, Karkas Özellikleri, Karaciger ve Plazma Çinko Konsantrasyonuna Etkileri. International Animal Nutrition Congress, 2000, 4-6 September, Isparta, Türkiye. 35. YI, Z., KORNEGAY, E.T. and DENBOW, D.M.: Supplemental Microbial Phytase Improves Zinc Utilization in Broilers. Poultry Sci., 1996, 75, 540-546. 36. ZAR, J. H.: Biostatistical Analysis, 4 th Edition. Prentice Hall, Upper Saddle River, New Jersey, 07458, USA, 1999.