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ANIMAL NUTRITION |

* Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2; and
Danisco Animal Nutrition, Marlborough, SN8 1AA, UK
| Abstract |
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Key Words: nutrient digestibility phytase pig performance wheat xylanase
| INTRODUCTION |
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The objective of this study was to determine the effect of supplementing wheat-based diets with phytase and xylanase alone or in combination on nutrient digestibility and performance of growing pigs.
| MATERIALS AND METHODS |
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Three experiments were conducted at the Animal Science Research Unit of the Department of Animal Science, University of Manitoba. The phytase and xylanase used in the experiments were Phyzyme XP (5,000 FTU/g) and Porzyme 9,300 (4,000 XU/g), respectively, from Danisco Animal Nutrition (Marlbourough, UK).
Exp. 1
This experiment was conducted to determine the effect of supplementing wheat-based diets with phytase and xylanase alone or in combination on apparent total tact digestibility (ATTD) of nutrients and on growth performance of pigs from 20 to 60 kg of BW. Sixty Cotswold pigs, obtained from the Glenlea Swine Research Unit, University of Manitoba, with BW of approximately 20 kg, were used in the experiment. Pigs were divided on the basis of BW and sex into 3 groups of 20 pigs each (10 barrows and 10 gilts), housed individually in pens (1.5 x 1.2 m) with smooth sides and plastic-covered expanded metal floors, and fed the experimental diets until they reached approximately 60 kg. Initial and final BW of all pigs were 19.9 ± 1.2 and 60.2 ± 2.4 (mean ± SD) kg, respectively. The length of time each pig received the experimental diets varied depending on its growth rate.
The 10 diets used in the experiment included a positive control diet (PC) and a negative control (NC) diet supplemented with phytase at 0, 250, or 500 FTU/kg and xylanase at 0, 2,000, or 4,000 XU/kg in a 3 x 3 factorial arrangement to give 9 treatment combinations (Tables 1
and 2
). The PC was formulated to meet or exceed the NRC (1998)
nutrient requirements for all nutrients for growing pigs weighing between 20 and 50 kg. The NC was similar to the PC except that Ca and available P contents were reduced by 16 and 30%, respectively, in an effort to maximize the response to enzyme supplementation. All diets were fed as mash.
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Exp. 2
This experiment was conducted to determine the effect of supplementing wheat-based diets with phytase and xylanase alone or in combination on apparent ileal digestibility (AID) of nutrients in growing pigs. Six crossbred barrows (Yorkshire-Landrace sow x Duroc boar), obtained from a local commercial swine herd (Genesus Genetics, Manitoba, Canada), with an average BW of 35.1 ± 1.6 (mean ± SD) kg were used in this experiment. Pigs were housed in adjustable metabolic crates with smooth, transparent plastic sides and a plastic-covered expanded metal floor. After a 7-d adaptation period, pigs were surgically fitted with a simple T-cannula at the distal ileum, as described by Nyachoti et al. (2002)
. After surgery, pigs were returned to the metabolic crates and allowed a 14-d recovery period. During this period, they were fed increasing amounts of a commercial grower diet twice daily and had unlimited access to water.
After the recovery period, pigs were fed 4 experimental diets, which consisted of a NC (same as that used in Exp. 1) or NC supplemented with phytase at 500 FTU/kg, xylanase at 4,000 XU/kg, or phytase at 500 FTU/kg plus xylanase at 4,000 XU/kg (Tables 1
and 2
). All diets contained 0.5% Cr2O3 as an indigestible marker and were fed as mash. The experiment was conducted according to a 4 x 4 Latin square design with 2 added columns. Each period consisted of 9 d; the first 7 d were for adaptation and the last 2 d for ileal digesta collection. Pigs were fed the diets at 2.6 times the maintenance energy requirement (ARC, 1981
) based on their BW at the beginning of each period. Daily feed allowance was offered in 2 equal portions at 0800 and 1530 h. Ileal digesta were collected continuously for 12 h from 0800 to 2000 h on d 8 and 9, as described by Nyachoti et al. (2002)
, and were stored at – 20 ° C until used for analysis.
Exp. 3
This experiment was conducted to determine the effect of supplementing phytase on ATTD of nutrients in growing-finishing pigs fed wheat-based diets in which most of the endogenous phytase in the wheat has been inactivated. Thirty-six Yorkshire growing-finishing barrows, obtained from a commercial swine herd in Manitoba (Iceman Genetics, Manitoba, Canada), and with an average BW of 55.5 ± 4.6 (mean ± SD) kg, were used in this experiment. Pigs were grouped based on BW into 3 groups, and housed individually in pens similar to those that were used in Exp. 1. After a 4-d acclimatization period to the new surroundings, during which they were fed a common commercial grower diet, pigs were randomly assigned to 4 experimental diets, which included PC, and a NC either unsupplemented or supplemented with phytase at 500 or 1,000 FTU/kg (Tables 1
and 2
). The PC was formulated to meet or exceed the NRC (1998)
nutrient requirements for all nutrients of growing pigs weighing between 50 and 80 kg. The NC was the same as the PC, except that Ca and available P contents were reduced by 20 and 35%, respectively, to maximize the response to enzyme supplementation. The wheat used in the diets was ground, prepelleted at 80 ° C, and crumpled before diet mixing to inactivate most of the endogenous phytase present in the wheat. This was done because, in a preliminary study in our laboratory, pelleting wheat at 80 ° C resulted in a reduction in endogenous phytase activity by 65% (unpublished data). All diets contained 0.2% Cr2O3 as an indigestible marker.
The experiment was conducted as a randomized complete block design, with the group of pigs considered as the block. The pigs were assigned to the experimental diets in such a manner that each diet was assigned to 3 pigs within each group, to give 9 replicates per diet. The experiment lasted for 2 wk. Representative fecal samples were collected from each pig during the last 3 d and stored frozen at – 20 ° C until used for analysis.
Sample Preparation and Chemical Analyses
Fecal samples for Exp. 1 were dried in an oven at 60 ° C for 4 d and pooled for each pig and period of collection (i.e., first and last 10 d of the experiment), finely ground in a grinder (CBG5 Smart Grind, Applica Consumer Products Inc., Shelton, CT), and thoroughly mixed for analysis. Ileal digesta samples for Exp. 2 were pooled for each pig and each period, homogenized in a blender (Waring Commercial, Torrington, CT), subsampled, freeze-dried, and ground as described previously for Exp. 1 for analysis. Fecal samples for Exp. 3 were dried as those for Exp. 1, pooled for each pig, and further prepared for analysis as described for Exp. 1. Diet samples for all the 3 experiments were similarly ground for analysis. All samples (diet, ileal digesta, and feces) were analyzed for DM, GE, CP, Ca, P, and Cr. Diet samples for Exp. 1 and 3 were additionally analyzed for NSP and enzymes (phytase and xylanase). Diets and ileal digesta for Exp. 2 were additionally analyzed for AA.
Dry matter was determined according to the method of the AOAC (1990)
and GE was determined using an adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline, IL). Crude protein (N x 6.25) was determined using a combustion analyzer (model CNS-2000; Leco Corp., St. Joseph, MI). Samples for Ca and P analyses were ashed and digested according to procedures described by the AOAC (1990)
and measured by inductively coupled plasma mass spectrometer (Varian Inc., Palo Alto, CA).
Diets and ileal digesta samples (Exp. 2) for AA analysis were prepared by acid hydrolysis according to the method of the AOAC (1984)
, and as modified by Mills et al. (1989)
. Briefly, about 100 mg of each sample was digested in 4 mL of 6 N HCl for 24 h at 110 ° C, followed by neutralization with 4 mL of 25% (wt/vol) NaOH and cooled to room temperature. The mixture was then equalized to 50 mL volume with sodium citrate buffer (pH 2.2) and analyzed using HPLC. Samples for analysis of sulfur containing AA (Met and Cys) were subjected to performic acid oxidation before acid hydrolysis. Tryptophan was not determined.
Samples for Cr analysis were ashed and digested according to procedures described by William et al. (1962)
and read on a Varian Inductively Coupled Plasma Mass Spectrometer (Varian Inc.). Nonstarch polysaccharides were determined by gas-liquid chromatography (component neutral sugars) and by colorimetry (uronic acids). The neutral sugars were analyzed as described by Englyst and Cummings (1988)
, with some modifications (Slominski and Campbell, 1990
), whereas uronic acids were determined using the procedure described by Scott (1979)
. The assay for phytase activity (the amount of the enzyme required to release 1 mmol of inorganic P per min from sodium phytate at 37 ° C) in the diets was conducted by Danisco Animal Nutrition (Marlborough, UK), as described by Engelen et al. (2001)
. Xylanase activity in feed was measured using a modified method based on the Megazyme xylanase assay kit (Megazyme International Ireland Ltd., Bray, Ireland).
Calculations and Statistical Analysis
Apparent ileal and total tract digestibility coefficients were calculated using the following equation:
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where Cd = the Cr2O3 concentration in the diet (% DM); Cf = the Cr2O3 concentration in the feces or ileal digesta (% DM); Nf = the nutrient concentration in the feces or ileal digesta (% DM); and Nd = the nutrient concentration in the diet (% DM). Digestible energy content of the positive and negative control diets was calculated using the following equation:
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Data from the 3 experiments were subjected to ANOVA using the MIXED procedure (SAS Inst. Inc., Cary, NC). In Exp. 1, the statistical model included the group (block effect), phytase, xylanase, and the interaction between phytase and xylanase. In Exp. 2, the statistical model included the period, animal, phytase, xylanase, and the interaction between phytase and xylanase, whereas in Exp. 3, it included the group (block effect) and phytase. In Exp. 1 and 3, specific contrasts were used to compare the PC diet or enzyme supplementation with the NC diet, and to compare the effect of level of phytase (250 vs. 500 FTU/kg in Exp. 1, and 500 vs. 1,000 FTU/kg in Exp. 3) and level of xylanase (2,000 vs. 4,000 XU/kg). In Exp. 2, specific contrasts were used to compare enzyme supplementation with the NC diet. In all the 3 experiments, differences were considered significant at P < 0.05.
| RESULTS |
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Analyzed chemical composition of the PC and NC diets and enzyme activities of the 10 dietary treatments are presented in Tables 1
and 2
, respectively. The analyzed values of CP and total P were similar to calculated values, whereas those of DE and Ca were greater than calculated values in Table 1
. The PC and NC diets were similar in NSP and endogenous phytase and xylanase activities. The addition of phytase and xylanase enzymes in diets generally resulted in increased activities of the respective enzymes by margins similar to those that were anticipated except for diets 6, 9, and 10 whose xylanase activity was increased by a much greater margin than anticipated after adding the xylanase enzyme (Table 2
). The ADFI, ADG, and G:F were not affected by dietary treatment (Table 3
).
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The effects of dietary treatment on AID measurements are presented in Table 5
. Phytase and xylanase interacted antagonistically (P < 0.05) on Lys, Ala, Asp, and Gly, and tended to interact antagonistically (P < 0.10) on His, Leu, Phe, Thr, Val, Ser, and Tyr. The digestibility values of these AA when the enzymes were supplemented individually were similar to the digestibility values when the enzymes were supplemented in a combination. Phytase supplementation increased (P < 0.05) the AID of Ca and P, but did not affect DE, and the AID of DM and CP. Phytase supplementation numerically improved the AID of all AA. Xylanase supplementation did not affect ileal DE, or the AID of DM, and P. However, xylanase increased (P < 0.05) the AID of Lys, Leu, Phe, Thr, Gly, and Ser, and tended to increase (P < 0.10) the AID CP, Ca, Arg, Ala, and Asp.
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Analyzed chemical composition of the PC and NC diets is presented in Table 1
. The analyzed values of DE, CP, Ca, and total P were greater than the calculated values in Table 1
. Data for DE and ATTD measurements are presented in Table 6
. The PC was greater in ATTD of P (P < 0.05) and Ca (P = 0.068) than NC. The 2 diets were, however, similar in ATTD of all other components measured in this experiment. The DE and ATTD of CP and Ca were not affected by phytase supplementation. Phytase supplementation increased ATTD of DM and P (P < 0.05) at both levels, but there was no further improvement at 1,000 FTU/kg relative to the 500 FTU/kg level.
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| DISCUSSION |
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The ATTD of components measured in the current study were generally greater for the basal diets in Exp. 3 than Exp. 1. This could be attributed to increased availability of nutrients for digestion after pelleting, which results in cell and starch granule rupture, thereby releasing the nutrients (ODoherty et al., 2001
). It could also be attributed to the older age of the pigs that were used in Exp. 3 compared with Exp. 1. Reports have indicated that the digestive capacity of animals increases with age due to an increase in mucosal surface area (Iji et al., 2001
). The ATTD of P for the PC diet in Exp. 3 was greater than that for the NC. The ATTD of P for the PC diet in Exp. 1 was also numerically greater than that for the NC. Adeola et al. (2004)
have also reported greater ATTD of P for a P-adequate diet compared with P inadequate diet, and they attributed it to the greater concentration of inorganic (available) P in the PC diet than in the NC. In contrast, the ATTD of DM (20 kg of BW) and Ca (20 and 60 kg of BW) digestibilities for the PC diet in Exp. 1 were lower than the NC. The greater Ca digestibility in the NC diet could be explained by lower Ca content in NC than in the PC diet, which was adequate in Ca. Low dietary Ca concentration has been shown to result in increased efficiency of Ca absorption from the gastrointestinal tract in an effort to maintain normal plasma Ca level (Weaver et al., 1996
). Traylor et al. (2001)
have also reported increased Ca digestibility in growing pigs due to a reduction in dietary Ca concentration.
Several studies have shown that phytase supplementation results in increased digestibility of P. Adeola et al. (2004)
reported that phytase supplementation at 250 FTU/kg to corn-based diets increased ATTD of P in growing pigs by at least 20%, whereas Kim et al. (2005b)
observed that ATTD of P increased by 35% after supplementation of wheat-based diets for weaner pigs with phytase at 500 FTU/kg. Similarly, Radcliffe et al. (2006)
reported that AID of P in growing pigs increased by 17% after supplementing a corn-based diet with phytase at 500 FTU/kg. Results of the current study are consistent with findings of these studies. In the current study, however, an increase in phytase supplementation from 250 to 500 FTU/kg did not result in a significant increase in ATTD of P at 60 kg in Exp. 1. Harper et al. (1997)
similarly reported a nonsignificant increase in ATTD of P in pigs heavier than 50 kg due to increasing phytase supplementation level from 250 to 500 FTU/kg. This lack of effect of increasing the level of supplemental phytase on P digestibility in pigs heavier than 50 kg compared with those lighter than 50 kg might be due to the low efficiency of absorption of P in the gastrointestinal tract due to their decreased dietary requirement of P. The efficiency of absorption of P has been shown to be inversely related to dietary available P concentration (Hattenhauer et al., 1999
).
Supplementation of phytase at 500 FTU/kg to the NC diet resulted in similar improvement in ATTD of P in Exp. 1 (at 60 kg of BW) and 3 (15.3 vs. 14.5 percentage units), which was unexpected. It had been assumed that phytase supplementation to the NC diet in Exp. 3 compared with Exp. 1 would result in greater improvement in P digestibility because (i) available P in NC diet in Exp. 3 had been greatly reduced (due to addition of small amount of corn) than that used in Exp. 1 (0.1 vs. 0.07 percentage units); and (ii) wheat used in Exp. 3 had been pelleted at 80 ° C, which resulted in decreased endogenous phytase activity in PC and NC diets used this experiment compared with in Exp. 1 (198 and 227 FTU/kg vs. 717 and 690 FTU/kg, respectively). This lack of difference in improvement of ATTD of P in the 2 experiments due to phytase supplementation could be attributed to greater P digestibility (40.0 vs. 22.8%) for the NC diet in Exp. 3 than Exp. 1. The response to phytase supplementation with regard to P digestibility has been shown to be high when the P digestibility in the basal diet is low (Johnston et al., 2004
).
Phytic acid in its natural state in feedstuffs is complexed with minerals and protein. In the stomach and small intestine, PA has potential to complex with positively charged nutrients and endogenous enzymes that are involved in nutrient digestion, thereby reducing nutrient digestibility (Lenis and Jongbloed, 1999
; Maenz, 2001
; Cowieson et al., 2004
). Thus, by hydrolyzing PA, phytase is not only expected to increase the digestibility of P, but of other nutrients as well. In the current study, phytase supplementation increased both AID (Exp. 2) and ATTD (Exp. 1) of Ca, which is in agreement with results of Johnston et al. (2004)
who reported improved AID and ATTD of Ca in growing-finishing pigs due to phytase supplementation. Phytase supplementation also numerically increased the AID of AA in Exp. 2, which is in agreement with the results of Traylor et al. (2001)
and Omogbenigun et al. (2003)
, who reported a numerical increase in AID of AA in pigs due to phytase supplementation. The ATTD of Ca in Exp. 3 was, however, unaffected by phytase supplementation. Also, increasing the level of phytase from 250 to 500 FTU/kg in Exp. 1 did not result in significant increase in ATTD of Ca. Calcium absorption in pigs has been reported to be greater at the colon than at the ileal level (Liu et al., 2000
). Therefore, the lack of an effect of phytase on ATTD of Ca in Exp. 3 could be due to increased digestibility of Ca in the large intestine of pigs used in Exp. 3 due to increased hindgut fermentation capacity. An increase in digesta fermentation in the hindgut can result in increased production of volatile fatty acids, which in turn can acidify the digesta and enhance solubility and hence absorption of Ca (Kruger et al., 2003
). The increased absorption in the hind-gut can mask the actual effect of phytase on Ca digestibility as demonstrated by the results of Radcliffe et al. (2006)
who reported increased Ca digestibility at the ileal but not fecal level of pigs heavier than 48 kg due to phytase supplementation. The lack of effect of increasing the level of phytase supplementation from 250 to 500 FTU/kg on ATTD of Ca in Exp. 1 could be due to the increased level of endogenous phytase in wheat that was used in this experiment. In the gastrointestinal tract, PA normally exerts its negative effect on mineral digestibility by binding them in the small intestine (Lenis and Jongbloed, 1999
). Wheat phytase is capable of hydrolyzing PA into partially dephosphorylated PA products (Schlemmer et al., 2001
) that have a decreased capacity to bind nutrients in the small intestine (Cowieson et al., 2006
). Thus, it is possible that the endogenous phytase in the basal diet plus supplemental phytase at 250 FTU/kg were adequate for hydrolysis of PA to partially dephosphorylated PA products that have low capacity to bind Ca.
Nutrient utilization in wheat-based diets is also limited by the presence of NSP in the wheat cell wall, which reduces the availability of nutrients in wheat for use by the animal by encapsulation (Kim et al., 2005a
). Apart from limiting nutrient availability for digestion, NSP can adsorb endogenous enzymes in the gastrointestinal tract (Silva and Smithhard, 2002
), thereby resulting in their increased secretion through a negative feedback mechanism (Wang et al., 2005
). The arabinoxylans can also increase the endogenous N losses by promoting the growth of microorganisms in small intestine, which utilizes endogenous AA, resulting in their reduced (endogenous AA) reabsorption (Bartelt et al., 2002
). In the current study, xylanase supplementation at 4,000 XU increased the AID of Ca and AA (Exp. 2) and ATTD of Ca at 20 kg of BW (Exp. 1), and marginally increased ATTD of energy at 60 kg of BW (Exp. 1). In wheat, Ca is highly associated with arabinoxylans (Frolich and Asp, 1985
). Thus, the increased digestibility of Ca resulting from xylanase supplementation could have been due to its increased availability after the hydrolysis of arabinoxylans by xylanase. The improved AID of AA could similarly be due to their increased availability of amino acids that are highly associated with arabinoxylans. The improved AID of AA could also be attributed to reduced secretion of endogenous AA due to hydrolysis of arabinoxylans by xylanase.
In wheat, both PA and arabinoxylans are concentrated in aleurone cells (Joyce et al., 2005
), and thus the presence of arabinoxylans in the walls of these cells can limit the accessibility of phytase to PA. Therefore, it was hypothesized that phytase and xylanase could act synergistically in improving nutrient digestibility because xylanase can hydrolyze arabinoxylans to release PA for phytase action. In the current study, however, phytase and xylanase did not synergistically interact on any of the response criteria measured, which might have been due to low reduction of available P and high endogenous phytase activity in the NC diet to which the enzymes were supplemented.
In conclusion, phytase supplementation to wheat-based diets for growing pigs improved P and Ca digestibilities, whereas xylanase supplementation increased Ca digestibility and apparent AA digestibility, but phytase and xylanase did not affect growth performance. Furthermore, there was no synergistic interaction between phytase and xylanse, which was probably due to high content of available P and endogenous phytase activity in the basal diets. Thus, xylanase and phytase may not show significant interaction in high endogenous phytase- and high digestible-wheat-based diets for pigs.
| Footnotes |
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2 Corresponding author: martin_nyachoti{at}umanitoba.ca
Received for publication January 8, 2007. Accepted for publication January 7, 2008.
| LITERATURE CITED |
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This article has been cited by other articles:
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J. S. Sands, D. Ragland, R. N. Dilger, and O. Adeola Responses of pigs to Aspergillus niger phytase supplementation of low-protein or high-phytin diets J Anim Sci, August 1, 2009; 87(8): 2581 - 2589. [Abstract] [Full Text] [PDF] |
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