EFFECT OF PHYTASE SUPPLEMENTATION TO DIETS FOR WEANLING PIGS ON THE UTILIZATION OF PHOSPHORUS  AND CALCIUM

Sheng Fa Liao, Willem C. Sauer, Arie Kies, Miguel Cervantes, John K. Htoo and Jim Ming He

Shengfa Liao. Ph.D in Animal Nutrition, University of Alberta, Canada. Research Associate, University of Kentucky, USA. e-mail: Shengfa.Liao@uky.edu

Willem C. Sauer. Ph.D. in Animal Nutrition. Emeritus Professor, University of Alberta, Canada. e-mail: willem.sauer@afhe.ualberta.ca

Arie Kies. MSc. in Non Ruminant Nutrition. DSM Food Specialties, Delft, The Netherlands.

Miguel Cervantes. Zoothecnical Engineer. Ph.D in Non Ruminant Nutrition, University of Kentucky, USA. Professor, Instituto de Ciencias Agrícolas, Universidad Autónoma de Baja California, México (ICA-UABC). Address: Lago Reindeer Nº 933, Jardines del Lago, Mexicali, B.C., México. Miguel_Cervantes@uabc.mx.

John K. Htoo. Ph.D., in Non Ruminant Nutrition, Research Associate, University of Alberta, Canada.

Jim Ming He. Ph.D in Non Ruminant Nutrition, Technischen Universität München-Weihenstephan, Germany. Research Associate, University of Alberta, Canada.

Summary

Four experiments were conducted with weanling pigs to determine the effect of phytase supplementation to four diet types on the apparent total tract digestibility and retention of P and Ca. Phytase was supplemented at rates of 0, 500, and 1000FTU/kg to the four diets. A 20% crude protein (CP) corn-soybean meal diet was used in Exp. 1; a 20% CP wheat-soybean meal diet in Exp. 2; a 20% CP wheat-soybean meal-canola meal diet in Exp. 3; and a 19% CP barley-peas-canola meal diet in Exp. 4. Six barrows were used in each experiment and fed the basal and phytase-supplemented diets according to a repeated 3´3 Latin square design. Each experimental period comprised 14 days. The piglets were fed at 08:00 and 20:00 daily equal amounts of each meal at a daily rate of at least 2.4 times the maintnance requirement for metabolizable energy. Feces and urine were collected from 08:00 on day 8 until 08:00 on day 12 of each experimental period. The results show that phytase supplementation, even to diets supplemented with inorganic P, increases the utilization of P and decreases the excretion in feces. The response of phytase on the utilization of Ca varies with diet composition and is dependent on the dietary Ca content. Phytase supplementation at a rate of 1000 compared to 500FTU/kg diet did not further improve the utilization of P and Ca.

EFECTO DE LA SUPLEMENTACIÓN DE FITASA EN DIETAS PARA CERDOS RECIÉN DESTETADOS SOBRE LA UTILIZACIÓN DE FÓSFORO Y CALCIO

Sheng Fa Liao, Willem C. Sauer, Arie Kies, Miguel Cervantes, John K. Htoo y Jim Ming He

Resumen

Se realizaron cuatro experimentos con cerdos destetados para determinar el efecto de la adición de fitasa a cuatro tipos de dietas en la digestibilidad total aparente y retención de P y Ca. La fitasa se adicionó en 0, 500 y 1000 FTU/kg de las cuatro dietas. En el Exp. 1 se utilizó una dieta maíz-pasta de soya, 20% proteína cruda (PC); dieta trigo-pasta de soya, 20% CP en Exp. 2; dieta trigo-pasta de soya-pasta de canola, 20% CP en Exp. 3; y cebada-garbanzo-pasta de canola, 19% PC en Exp. 4. Se usaron 6 cerdos en cada experimento y se alimentaron con las dietas base o las adicionadas con fitasa de acuerdo con un diseño en cuadro latino 3´3 repetido. Cada periodo experimental consistió de 14 días. Los cerdos se alimentaron a las 08:00 y 20:00, en cantidades iguales cada comida, equivalentes al menos a 2,4 veces las necesidades diarias de energía metabolizable. Las heces y orina se colectaron de 08:00 del día 8 a las 08:00 del día 12 de cada periodo experimental. La adición de fitasa, aún a dietas adicionadas con P inorgánico, aumenta la digestibilidad de P y reduce su excreción en heces. La respuesta a fitasa en la utilización de Ca varía con la composición de la dieta y es dependiente de su contenido de Ca. La adición de 1000 FTU/kg dieta, comparado con la de 500 FTU/kg dieta no resultó en una mejora adicional en la utilización de P y Ca.

EFEITO DA FITASE SUPLEMENTAR EM DIETAS PARA SUÍNOS DESMAMADOS COM A UTILIZAÇÃO DE FÓSFORO E CÁLCIO

Sheng Fa Liao, Willem C. Sauer, Arie Kies, Miguel Cervantes, John K. Htoo e Jim Ming He

Resumo

Realizaram-se quatro experimentos com suínos desmamados para determinar o efeito da adição de fitase a quatro tipos de dietas na digestibilidade total aparente e retenção de P e Ca. A fitase se adicionou em 0, 500 e 1000 FTU/kg das quatro dietas. No Exp. 1 utilizou- se uma dieta milho-pasta de soja, 20% proteína crua (PC); dieta trigo-pasta de soja, 20% CP no Exp. 2; dieta trigo-pasta de soja-pasta de canola, 20% CP no Exp. 3; e cevada-grão-de-bico-pasta de canola, 19% PC no Exp. 4. Usaram-se 6 suínos em cada experimento e se alimentaram com as dietas base ou as adicionadas com fitase de acordo com um desenho em quadro latino 3´3 repetido. Cada período experimental consistiu de 14 dias. Os suínos se alimentaram às 08hs e 20hs em quantidades iguais cada comida, equivalentes ao menos a 2,4 vezes as necessidades diárias de energia metabolizável. As fezes e urina se recolheram das 08hs do dia 8 às 08hs do dia 12 de cada período experimental. A adição de fitase, mesmo a dietas adicionadas com P inorgânico, aumenta a digestibilidade de P e reduz sua excreção nas fezes. A resposta a fitase na utilização de Ca varia com a composição da dieta e é dependente do seu conteúdo de Ca. A adição de 1000 FTU/kg dieta, comparado com a de 500 FTU/kg dieta não resultou em uma melhora adicional na utilização de P e Ca.

KEYWORDS / Calcium / Digestibility / Phosphorus / Phytase / Weanling Pigs /

Received: 07/11/2005. Modified: 01/02/2006. Accepted: 01/16/2006.

 

Introduction

The very limited ability of pigs to utilize phytate P poses three problems. The first problem is environmental pollution resulting from excretion of a large proportion of P into manure. Concerns over this issue have been globally expressed. The second problem relates to the supplementation of inorganic P, which is relatively expensive, to swine diets. Often, inorganic P is over supplied to ensure that the requirement for available P is met, which makes the first problem even worse (NRC, 1998). The third problem relates to the ability of phytate, including phytic acid, to complex with other dietary nutrients such as Ca (Liao et al., 2002).

Phytase is an enzyme that catalyzes the hydrolysis of orthophosphate groups from phytate molecules. Pigs lack this enzyme. It has been shown in numerous investigations that supplementation of microbial phytase to swine diets can improve the utilization of phytate P (Cromwell et al., 1993; 1995; Lei et al., 1993a, b). The optimum supplementation rate appears to be 500FTU/kg diet, although a maximum response can be achieved at 1000FTU/kg diet (Canh, 1995; Jongbloed et al., 2000). Nevertheless, nearly all studies were conducted with corn-soybean meal diets that were not supplemented with inorganic P. Therefore, further studies with corn-soybean meal and other diets containing different ingredients supplemented with inorganic P, as is often practiced, are warranted. The effect of phytase supplementation on the utilization of Ca also needs to be determined as this was investigated in only a few studies, and the results were not always consistent (Kemme et al., 1999; Traylor et al., 2001).

This study was carried out to determine the effect of phytase supplementation on the utilization of P and Ca in weanling pigs fed different starter diets supplemented with inorganic P. Another objective of the study was to determine if a higher rate of supplementation (1000 vs. 500FTU/kg diet) can further improve the utilization of P and Ca in these diets.

Experimental Procedures

Experiments and diets

Four experiments were carried out with weanling pigs. In each experiment, the piglets were fed a basal diet consisting of commonly used ingredients (Table I). To each basal diet, Aspergillus niger phytase (Natuphos®, BASF AG, Ludwigshaven, Germany) was supplemented at rates of 500 and 1000FTU/kg. One FTU is defined as the quantity of enzyme that liberates 1µM of orthophosphate per min from 5.1mM Na-phytate at pH 5.5 and at 37ºC (Engelen et al., 2001). The diets were supplemented with a mixture of mono- and dicalcium phosphate to meet the NRC (1998) standard for available P, which is 0.32% for weanling pigs.

Canola oil was included in the diets to increase the digestible energy content to the level recommended by NRC (1998) and synthetic aminoacids were supplemented to meet the NRC (1998) standards. Vitamins and minerals were supplemented to meet or exceed NRC (1998) standards. Chromic oxide was included in the diet at a rate of 0.30% as a digestibility marker.

As shown in Table I, the major ingredients of the basal diet in Exp. 1 were corn and soybean meal. In Exp. 2, the major ingredients were wheat and soybean meal. In Exp. 3, the major ingredients were wheat, soybean meal and canola meal. In Exp. 4, the major ingredients were barley, peas and canola meal. The major ingredients were ground through a 2mm mesh sieve prior to diet formulation. All diets were fed in the form of mash. Water was freely available from a low-pressure drinking nipple.

Animal trial procedures

For each of the four experiments, six PIC (Pig Improvement Co., Airdrie, Alberta, Canada) barrows (Camborough×Canabrid), weaned at 3 weeks of age, were obtained from the University of Alberta Swine Research Unit. The barrows were housed individually in metabolism crates (82, 124 and 76cm of height, length and width, respectively) in a barn, where the temperature was maintained at 25 to 28ºC. On days 6 and 7 after weaning and adjustment to the crates, each piglet was fitted with a simple T-cannula at the distal ileum, approximately 5cm from the ileo-cecal sphincter. Usually, intact pigs are used for studying the apparent total-tract digestibilities (ATTD) of minerals. In this trial, however, the pigs were cannulated because they were also used for the determination of ileal digestibilities of crude protein and amino acids (Liao et al., 2005). It needs to be pointed out that cannulation has little effect on the ATTD of amino acids (Sauer and Ozimek, 1986). Therefore, it is unlikely that cannulation will affect the ATTD of P and Ca.

Following a 1 week recuperation period after surgery, the piglets were fed the experimental diets according to a repeated 3´3 Latin Square design. Each experimental period comprised 14 days. In Exp. 1 and 2, the daily meal allowances were fed to the piglets at a rate of 2.4 times the maintenance requirement for metabolizable energy (ME; i.e., 418.4 kJ/BWkg0.75), based on the individual BW of the pigs, which was determined at the beginning of each experimental period. In Exp. 3 and 4, the piglets were fed at a rate of 5% (wt/wt) of BW. In Exp. 3, the feed intake was equivalent to 2.7, 2.9, and 3.0 times the maintenance requirement for ME during periods 1, 2, and 3, respectively. In Exp. 4, the feed intake was equivalent to 2.8, 3.0, and 3.2 times the maintenance requirement for ME during periods 1, 2, and 3, respectively. Before the initiation of the Exp. 3 and 4, the piglets seemed very hungry. Therefore, the feed intake was increased. The meal allowances were offered twice daily at 08:00 and 20:00, equal amounts each meal.

The average body weights of the piglets were 7.6, 8.6, 7.3, and 8.0kg at the initiation of Exp. 1, 2, 3, and 4, respectively. The average body weight of the piglets at the conclusion of Exp. 1, 2, 3, and 4 were 15.1, 17.7, 16.3, and 16.8kg, respectively.

The animals used in this study were cared for in accordance with the guidelines established in CCAC (1993), and the Animal Care Committee of the Faculty of Agriculture, Forestry and Home Economics, University of Alberta, Canada, approved the experimental proposal.

Sample collection and chemical analysis

Samples of the major feed ingredients were taken prior to diet formulation. Samples of the diets were taken during the time the meal allowances were prepared. The collection of feces and urine were initiated at 08:00 on day 8 of each experimental period and continued for 96 consecutive hours. Feces were collected by aid of colostomy bags (Stomahesive Wafer, Sur-Fit Natura, ConvaTec, Princeton, NJ, USA). The area around the anus was shaved and the bags were attached by aid of a medical adhesive glue (Hollister, Libertyville, IL, USA). The bags were replaced every 4 to 8h depending on the amount of feces collected. Feces were frozen at -28ºC immediately after collection. Concentrated sulfuric acid (5ml) was added to each urine flask. Urine, collected through glass wool, were stored at -4ºC immediately after collection, then filtered through triple layers of medical gauze and pooled for each pig in each experimental period and frozen at -28ºC. Urine samples were collected three times daily, at 08:00, 14:00, and 20:00.

Before chemical analyses, feces were air-dried and pooled, leaving one sample for each pig in each experimental period. Samples of diets and feces were ground to pass a 0.5mm mesh screen in a Thomas-Wiley Laboratory mill (Arthur H. Thomas Co., Philadelphia, PA, USA). Samples of urine were first filtered through Whatman #2 filter paper, and then air-dried at 60ºC.

The dry matter content in the diets and feces were determined according to AOAC (2000) official methods 930.15. The crude protein content (N×6.25) in the diet was measured with an FP-428 Nitrogen Determinator (Leco® Corporation, St. Joseph, MI, USA). The P and Ca contents in diets, feces, and urine were determined according to AOAC (2000) methods 965.17 and 968.08, respectively. The phytate P contents in the diets were analyzed according to Haug and Lantzch (1983). The phytase activities of the diets were analyzed with an enzymatic procedure according to AOAC (2000) method 2000.12 (Engelen et al., 1994, 2001). Chromic oxide was determined with a photometric procedure according to Fenton and Fenton (1979). Analyses of diets were carried out in triplicate; analyses of feces and urine in duplicate.

Digestibility calculations and statistical analysis

The ATTD of P and Ca were calculated by using the equation:

DD = 100% - [(AF × ID) / (AD × IF)] × 100%

where DD: ATTD of P or Ca in the assay diet (%), AF: P or Ca concentration in feces (%), ID: chromic oxide concentration in the assay diet (%), AD: P or Ca concentration in the assay diet (%), IF: chromic oxide concentration in feces (%). The urinary excretions of P and Ca were also measured to determine their balance values.

The ATTD and balance values were subjected to statistical analysis using the GLM Procedure of SAS (1988) with the following linear model:

Yijk = µ + Ti + Pj + Ak + eijk

where Yijk: an ATTD or balance value; m: overall mean of the ATTD or balance values; Ti: fixed effect of phytase treatment and i= 1, 2, 3; Pj: random effect of experimental periods and j= 1, 2, 3; Ak: random effect of animals and k= 1, 2, 3, 4, 5, 6; eijk: random experimental error with N (0, s2). Two orthogonal contrasts were constructed to test the effect of phytase supplementation to the basal diet (C1= 0 vs. 500 + 1000FTU/kg diet) and the phytase supplementation level (C2= 500 vs. 1000FTU/kg diet). Probability levels of P£0.05 and 0.05<P£0.10 were defined as significant differences and tendencies, respectively.

Results

All piglets remained healthy and usually consumed their meal allowances within 30min after feeding. The ADG of the piglets in Exp. 1, 2, 3 and 4 were 178, 221, 216 and 211g/day, respectively. Postmortem examinations conducted at the conclusion of each experiment revealed no adhesions or any other intestinal abnormalities.

The chemical composition of the four basal diets are presented in Table II. The phytate P contents of the diets ranged between 0.16 and 0.21%, while the intrinsic phytase activities ranged between 53 and 419FTU/kg. The phytase activities of the phytase-supplemented diets are also presented in Table II. It should be noted that the phytase activities in the basal diet of Exp. 4, supplemented with 500 (815FTU/kg) and 1000 FTU/kg (924FTU/kg) were similar. It is likely that an error was made in mixing the diet supplemented with 1000FTU/kg. The content of CP in diet 4 was 18.9% and lower than in the other diets which ranged from 20.3 to 21.3%. Diet 4 included synthetic methionine, threonine, and tryptophan (Table I).

Phosphorus balance

Phytase supplementation to all diets improved the ATTD of P (P<0.01; Table III). The magnitudes of improvement ranged from 6.5 to 9.5 percentage units (pu) for the corn-soybean meal diet, 9.1 to 12.7pu for the wheat-soybean meal diet, 8.5 to 11.2pu for the wheat-soybean meal-canola meal diet, and 11.8 to 11.9pu for the barley-peas-canola meal diet. Except for the wheat-soybean meal diet, there were no differences (P>0.10) in the ATTD of P between the two rates of phytase supplementation. The ATTD of P in the wheat-soybean meal diet was 3.6pu higher (P<0.01) when phytase was supplemented at a rate of 1000 compared to 500FTU/kg.

Phytase supplementation to all diets decreased (P<0.05) the daily fecal output of P (Table III). The decreases in the fecal output ranged from 0.12 (9.0%) to 0.25g (18.7%) for the corn-soybean meal diet, from 0.24 (20.7%) to 0.35g (30.2%) for the wheat-soybean meal diet, from 0.22 (10.9%) to 0.42g (20.8%) for the wheat-soybean meal-canola meal diet, and from 0.48 (26.5%) to 0.51g (28.2%) for the barley-soybean meal-canola meal diet. There was a larger decrease (P<0.05) in the fecal output of P for the corn-soybean meal and wheat-soybean meal diets when phytase was supplemented at a rate of 1000 compared to 500FTU/kg.

The daily urinary output of P (Table III) increased upon phytase supplementation to the corn-soybean meal (P < 0.05) but not to the other diets (P > 0.10). Phytase supplementation to the wheat-soybean meal or the wheat-soybean meal-canola meal diet, at a rate of 1,000 compared to 500 FTU/kg, tended to increase the urinary output of P (P < 0.10).

The retention of P increased (P<0.05) upon phytase supplementation to all diets. The average magnitude of increases were 7.0pu for the corn-soybean meal diet, 9.9pu for the wheat-soybean meal diet, 9.3pu for the wheat-soybean meal-canola meal diet, and 11.4pu for the barley-peas-canola meal diet. Phytase supplementation to the wheat-soybean meal diet, at the rate of 1000 compared to 500FTU/kg, tended to increase (P<0.10) the retention of P.

Calcium balance

As shown in Table IV, phytase supplementation increased the ATTD of Ca in the wheat-soybean meal diet (P<0.01) and the barley-peas-canola meal diet (P<0.05). The magnitudes of increase (P<0.05) ranged from 8.7 to 8.8pu for the wheat-soybean meal diet, and from 8.6 to 8.9pu for the barley-peas-canola meal diet. The improvements in the ATTD of Ca in the corn-soybean meal diet and the wheat-soybean meal-canola meal diet upon phytase supplementation were not significant (P>0.10). As well, in all experiments there were no differences (P>0.10) in the ATTD of Ca between the two rates of phytase supplementation.

The daily fecal output of Ca (Table IV) decreased only when phytase was supplemented to the wheat-soybean meal (P<0.01) and the barley-peas-canola meal (P<0.05) diets. The decreases ranged from 0.32 (28.8%) to 0.35g (31.5%) for the wheat-soybean meal diet and from 0.35 (26.7%) to 0.3 g (28.2%) for the barley-peas-canola meal diet. There were no differences (P>0.10) in the fecal output of Ca between the two rates of phytase supplementation to all diets.

There were no differences (P>0.10) in the daily urinary output of Ca between the phytase-supplemented and basal diets (Table IV). Likewise, there were no differences between the two rates of phytase supplementation (P>0.10).

The retention of Ca increased upon the supplementation of phytase to the wheat-soybean meal (P<0.01) and the barley-peas-canola meal (P<0.05) diets. There was no difference (P>0.10) in the retention between the two rates of supplementation. For the wheat-soybean meal-canola meal diet, there was a tendency (P<0.10) towards an increase in the retention of Ca.

Discussion

Commercial preparations of microbial phytase were initially developed to increase the bioavailability of phytate P in feedstuffs of plant origin for nonruminants in order to reduce the animal reliance on inorganic P supplementation, decrease the P output in manure, and alleviate P pollution to the environment. Previous studies, mainly with growing and finishing pigs fed corn-soybean meal diets that were not supplemented with inorganic P, have shown that supplementation can achieve these objectives (e.g., Cromwell et al., 1993; Kemme et al., 1999). Studies with weanling pigs, also fed corn-soybean meal diets that were not supplemented with P, have also shown that the ATTD of P was increased upon phytase supplementation (Lei et al., 1993a; Young et al., 1993). The supplementation of phytase to the four diets improved the ATTD of P by 6.5 to 12.7pu, equivalent to 13.3 to 22.4% (Table III). The magnitude of improvements in the ATTD of P were smaller in this study than in other studies reported in the literature. The smaller improvements can be explained by the relatively high concentration of P in the diets, ranging from 0.59 to 0.76% (Table II) as inorganic P was supplemented. Lei et al. (1993a) reported that phytase supplementation to a corn-soybean meal diet low in total P (0.32%) improved the ATTD of P from 46.4 to 69.0%. On the assumption that P from mono-dicalcium phosphate is completely available (NRC, 1998), the improvements in the ATTD of P in this study would be 23.5, 27.5, 31.9, and 32.0pu in Exp. 1, 2, 3, and 4, respectively. These values are more in the range of improvements reported in other studies (Young et al., 1993; Näsi and Helander, 1994).

The daily outputs of P in feces were reduced by 13.8, 25.4, 15.8 and 27.3% in Exp. 1, 2, 3 and 4, respectively (Table III). The output of P in urine was very small compared to feces, irrespective of phytase supplementation. Therefore, with an increase in the ATTD of P there is a corresponding increase in the retention of P. Indirectly, these results may also imply that the NRC (1998) standard for available P for weanling pigs, which is 0.32%, is too low.

Phytate residues or phytic acid may bind polyvalent cations, such as Ca, forming insoluble phytate-mineral complexes, and rendering these poorly available to nonruminants (Jongbloed et al., 2000). As was reviewed by Liao et al. (2002), although Ca among several mineral elements has the lowest binding affinity to phytic acid under in vitro conditions, the greatest impact of phytate on the bioavailability of minerals other than P is on Ca because of its relatively high dietary content. A high intake of phytate can reduce Ca absorption and utilization for bone formation in rats, puppies, chickens and humans (Reddy et al., 1989). Oberleas et al. (1962) reported that the supplementation of phytic acid to diets for pigs caused a depression in growth rate. Simons et al. (1990), in studies with growing pigs fed a tapioca meal-based diet, were the first to report an increase in the ATTD of Ca upon phytase supplementation. Näsi (1990) reported that both the ATTD and retention of Ca were improved upon phytase supplementation to a corn-soybean meal diet in studies with growing and finishing pigs.

Studies with weanling pigs have also shown that phytase supplementation can improve the ATTD and/or retention of Ca. For example, Lei et al. (1993a) reported that the excretion of Ca in feces was reduced by 52% in weanling pigs fed a corn-soybean meal diet. Other studies with young pigs have shown no effect of phytase supplementation on the ATTD and/or retention of Ca (e.g., Yi et al., 1996). In the present study, supplementation of phytase increased the ATTD of Ca in the wheat-soybean meal and in the barley-peas-canola meal diet, but not in the other diets (Table IV). Correspondingly, the retention of Ca was also improved in the wheat-soybean meal and barley-peas-canola meal diets. These results suggest that a positive response to phytase supplementation may occur when the diets are relatively low in Ca. The dietary Ca levels were 0.84 and 0.70% in the wheat-soybean meal and barley-peas-canola meal diets, respectively. The Ca levels in the corn-soybean meal and wheat-soybean meal-canola meal diets were higher, namely 0.94 and 1.05%, respectively (Table II). Jongbloed et al. (1993) reported that the ATTD of Ca was not only related to the rate of phytase supplementation, but also to the dietary content.

Implications

Supplementation of microbial phytase to diets formulated with commonly used feed ingredients, even with inorganic P supplementation, improves the utilization and decreases the fecal excretion of P in weanling pigs. The effect of phytase supplementation on the utilization of Ca varies with diet composition. Phytase supplementation at a rate of 1000 compared to 500FTU/kg diet did not improve the utilization of P and Ca.

ACKNOLEDGEMENTS

Financial support was provided by DSM Food Specialties, Delft, The Netherlands, and the Alberta Livestock Industry Development Fund Ltd. and the Alberta Agricultural Research Institute, Canada. This work was presented in part at the 2003 Banff Pork Seminar, Banff, AB, Canada, and the 9th International Symposium on Digestive Physiology in Pigs, 2003, Banff, AB, Canada.

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