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ANIMAL NUTRITION |
* Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada;
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DSM Food Specialties, 2600 MA Delft, The Netherlands; and
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Instituto de Ciencias Agri 'colas, Universidad Auto 'noma de Baja California, Mexicali, Me 'xico 21100
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Abstract |
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 Top Abstract IntroductionExperimental ProceduresResultsDiscussionImplicationsLiterature Cited |
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Key Words: Amino Acids • Digestibility • Energy • Phytase • Piglets
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Introduction |
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TopAbstractIntroductionExperimental ProceduresResultsDiscussionImplicationsLiterature Cited |
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). Phytase catalyzes the removal of orthophosphate groups from phytate (Maga, 1982), allowing the bound nutrients to be released for utilization. The activity of intrinsic phytase in diets for pigs and activity of endogenous phytase in the digestive tract are not sufficient for efficient hydrolysis of phytate (Pointillart et al., 1987; Cromwell et al., 1993).
The supplementation of microbial phytase to swine diets can improve the digestibility and retention of P in pigs (e.g., Simons et al., 1990; Lei et al., 1993a,b). Supplementation of phytase to diets for pigs also improved the apparent ileal digestibilities (AID) of CP and some of the AA (Mroz et al., 1994; Kemme et al., 1999). Other studies showed no effect of phytase supplementation on the AID of CP and AA (Traylor et al., 2001; Sands, 2002). The lack of consistency in response to phytase supplementation was recently discussed by Adeola and Sands (2003), who cautioned against the use of any overly simplistic guidelines that ascribe an "AA response factor" to phytase supplementation.
The objective of this study was to determine whether there is a diet-dependent AA response factor to phytase supplementation in addition to the apparent total-tract digestibility (ATTD) of CP and the content of DE. Phytase was supplemented to different diets for weanling pigs formulated with commonly used feed ingredients.
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Experimental Procedures |
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TopAbstractIntroductionExperimental ProceduresResultsDiscussionImplicationsLiterature Cited |
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). To each basal diet, Aspergillus niger phytase (Natuphos, DSM Food Specialties, Delft, The Netherlands) was supplemented at rates of 500 and 1,000 FTU/kg (as-fed basis). One FTU is defined as the quantity of enzyme that liberates 1 µM of ortho-phosphate per min from 5.1 mM Na-phytate at pH 5.5 and at 37°C (Engelen et al., 2001). The diets were supplemented with a mixture of mono- and dicalcium phosphate to meet the NRC (1998) standards for available P, which is 0.32% for weanling pigs. Canola oil was included in the diets to increase the DE content to the level recommended by NRC (1998). The calculated ME contents (based on NRC [1998] recommendations) of the basal diets in Exp. 1, 2, 3, and 4 were 3,183, 3,240, 3,248, and 3,302 kcal/kg (as-fed basis), respectively. Synthetic AA were supplemented to diets in Exp. 4 to meet 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% of the diet as a digestibility marker.
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, 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, whereas 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 to pass a 2-mm mesh sieve before diet formulation. Before surgery and during the recuperation period, the piglets were fed ad libitum a starter diet containing 20% CP. All diets were fed in the form of a 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 x Canabrid), weaned at 3 wk of age, were obtained from the University of Alberta Swine Research Unit. The barrows were housed individually in metabolism crates (height = 82 cm; length = 124 cm; width = 76 cm) in a barn maintained at 25 to 28°C. On d 6 and 7 after weaning, each piglet was fitted with a simple T-cannula at the distal ileum, approximately 5 cm from the ileocecal sphincter. Detailed descriptions of cannula preparation, surgery, and pre- and postoperative care were previously given by Sauer (1976) and Li et al. (1993).
Following a 1-wk recuperation period after surgery, the piglets were fed the experimental diets according to a repeated 3 x 3 Latin square design. Each experimental period comprised 14 d. In Exp. 1 and 2, the piglets were fed at a rate of 2.4 times the maintenance requirement for ME (i.e., 100 kcal/BW0.75kg), based on the BW of each piglet, which was determined at the beginning of each experimental period. In Exp. 3 and 4, the piglets were fed at a rate of 5% 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, 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 0800 and 2000, equal amounts each meal.
The animals used in this study were cared for in accordance with the guidelines established by the CCAC (1993), and the Animal Care Committee of the Faculty of Agriculture, Forestry and Home Economics, University of Alberta approved the experimental proposal.
Sample Collection and Chemical Analyses
Samples of the major feed ingredients were taken before diet formulation. Samples of the diets were taken during the time the meal allowances were prepared. The collection of feces was initiated at 0800 on d 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). The bags were changed every 4 to 8 h, depending on the quantity of feces collected. Feces were frozen at –28°C immediately after collection. Ileal digesta were collected into a soft plastic tube (length, 15 cm; i.d., 4 cm) for 36 h: from 0800 to 2000 on d 12, 13, and 14. Before collection, 5 mL of 10% (vol/vol) formic acid solution was placed into each tube. The tube was attached to the barrel of the cannula with a rubber band and was removed and replaced as soon as it was nearly filled with digesta. Digesta were frozen at –28°C immediately after collection. Detailed procedures for collection of ileal digesta were previously described by Sauer (1976) and Li et al. (1993).
Before chemical analyses, feces and digesta were pooled leaving one sample for each pig in each experimental period. Feces were air-dried, and digesta were freeze-dried. The dried samples of feces and digesta, and samples of ingredients and diets, were ground to pass a 0.5-mm mesh screen in a Thomas-Wiley Laboratory Mill (Arthur H. Thomas Co., Philadelphia, PA).
Crude protein (N x 6.25) was measured with a Leco FP-428 nitrogen determinator (Leco Corp., St. Joseph, MI). Gross energy was determined with an AC-300 Leco automatic calorimeter. The P contents in the basal diets were determined spectrophotometrically by the molyb-dovanadate procedure according to procedure No. 965.17 (AOAC, 2000). The phytate P contents in the basal diets were analyzed according to procedures described by Haug and Lantzsch (1983). The phytase activities (FTU/kg diet) were analyzed with a colorimetric enzymatic procedure according to AOAC (2000) official method 2000.12 (Engelen et al., 1994, 2001). Chromic oxide was determined with a spectrophotometric procedure according to Fenton and Fenton (1979). Analyses of ingredients and diets were carried out in triplicate; analyses of feces and digesta were in duplicate.
For AA analyses, approximately 0.05 g of finely ground digesta (<0.1 mm) was weighed into 13 x 100 mm Pyrex culture tubes with Teflon-lined screw caps (VWR Canlab, Edmonton, Alberta, Canada), mixed with 3 mL of 6 N HCl, and hydrolyzed at 110°C for 24 h in an oven. The hydrolyzed samples were mixed with an internal standard, DL-amino-n-butyric acid, and centrifuged at 1,110 x g for 15 min at 4°C. The supernatant fraction of the sample was analyzed using a Varian 5000 HPLC system with a reverse-phase column and a Varian Fluorichrom detector (Varian Canada Inc., Mississauga, Canada), according to principles outlined by Jones and Gilligan (1983). The AA were derivatized with an o-phthaldialdehyde reagent solution. The mobile phase consisted of two solvents (A and B), with a flow rate of 1.1 ml/min. Solvent A was a 0.1 M sodium acetate (pH 7.2), methanol, and tetrahydrofuran in a ratio of 18 to 1; solvent B was pure methanol. Peaks were recorded and integrated using the Ezchrom Chromatography Data System (Version 4.2; Shimadzu Scientific Instruments Inc., Columbia, MD). The procedure was described in detail by Sedgwick et al. (1991). The sulfur-containing AA, proline, and tryptophan were not determined.
Digestibility Calculations and Statistical Analyses
The AID of CP and AA, and the ATTD of CP and GE were calculated by using the following equation:
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where DD is the apparent digestibility of a nutrient or energy in the assay diet (%), AF is the nutrient or energy concentration in ileal digesta or feces (%), ID is the indicator concentration in the assay diet (%), AD is the nutrient or energy concentration in the assay diet (%), and IF is the indicator concentration in ileal digesta or feces (%).
Based on the following linear model, the digestibility data were subjected to statistical analysis using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC):
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where Yijk is a digestibility value; µ is the overall mean of the digestibility values; Ti is the fixed effect of treatments; i = 1, 2, 3; Pj is the random effect of experimental periods; j = 1, 2, 3; Ak is the random effect of animals; k = 1, 2, 3, 4, 5, 6; and 
ijk is the residual experimental error with N (0, 
2). Two orthogonal contrasts were constructed to test the effect of phytase supplementation to the basal diet (C1 = 0 vs. 500 + 1,000 FTU/kg diet) and the phytase supplementation level (C2 = 500 vs. 1,000 FTU/kg diet). Probability levels of P 
0.05 and 0.05 < P 0.10 were defined as significant differences and tendencies, respectively.
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Results |
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TopAbstractIntroductionExperimental ProceduresResultsDiscussionImplicationsLiterature Cited |
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. The analyzed values of CP and AA in the diets were close to the values calculated based on the analyzed values in the ingredients. The phytate P contents of the basal diets ranged from 0.16 to 0.22%, whereas the intrinsic phytase activities ranged from 53 to 419 FTU/kg. The total phytase activities of the phytase-supplemented diets are also presented in Table 2. It should be noted that the phytase activities in the basal diet of Exp. 4, supplemented with 500 (815 FTU/kg) and 1,000 FTU/kg (924 FTU/kg), were similar. It is likely that an error was made in mixing the diet supplemented with 1,000 FTU/kg. The content of CP in the basal diet of Exp. 4 was 18.9% and lower than in the other diets, which ranged from 20.3 to 21.3%. The diet of Exp. 4 contained synthetic methionine, threonine, and tryptophan (Table 1).
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Apparent Ileal Digestibilities of Crude Protein and Amino Acids
The effect of phytase on the AID of CP and AA are presented in Tables 3 to 6. Phytase supplementation to the corn-soybean meal diet (Table 3, Exp. 1), the wheat-soybean meal diet (Table 4, Exp. 2), or the barley-peas-canola meal diet (Table 6, Exp. 4) did not affect the AID of CP and AA (P = 0.238 to 0.981). However, phytase supplementation to the wheat-soybean meal-canola meal diet resulted in significant increases or tendencies towards increases in the AID of CP and AA (Table 5, Exp. 3). The supplementation of phytase (average of 500 and 1,000 FTU/kg diet) increased the AID of arginine (P = 0.041), histidine (P = 0.012), phenylalanine (P = 0.048), threonine (P = 0.042), and valine (P = 0.045). Phytase supplementation also tended to increase the AID of CP (P = 0.079), leucine (P = 0.067), lysine (P = 0.066), alanine (P = 0.069), aspartic acid (P = 0.096), and tyrosine (P = 0.55). There was no effect (P = 0.622 to 0.983) of phytase supplementation level (500 vs. 1,000 FTU/kg diet) on the AID of AA.
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. There was a significant increase in the ATTD of CP with phytase supplementation in Exp. 2 (P = 0.006) and Exp. 3 (P = 0.021). The content of DE tended to increase in Exp. 3 (P = 0.067). There was no effect of phytase supplementation level on the ATTD of CP (0.820 > P > 0.166) or DE contents (0.699 > P > 0.178) in any of the experiments. As was expected, the ATTD of CP were higher than their corresponding AID for all diets, ranging from 6.7 to 12.0 percentage units (pu; Table 7 vs. Tables 3, 4, 5, and 6).
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Discussion |
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TopAbstractIntroductionExperimental ProceduresResultsDiscussionImplicationsLiterature Cited |
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pointed out that phytase supplementation does not consistently improve the digestibilities of CP and AA. On a theoretical basis, four mechanisms can be suggested for a possible negative effect of phytate on the AID of CP and AA: 1) inherent phytate-protein-AA complexes in feedstuffs; 2) de novo formation of phytate-protein complexes in the digestive tract; 3) de novo formation of phytate-AA complexes after digestion of dietary protein in the digestive tract; and 4) formation of complexes between phytate and proteolytic enzymes in the digestive tract (Kies et al., 1997; Selle et al., 2000). Conceptually, the extent to which phytase supplementation may increase the AID of CP/AA will be dependent, in part, on the proportion of protein-AA bound in phytate bonds. But, as was discussed by Kemme et al. (1999), there are many other factors that should be considered, namely the type and the solubility of protein, in situ pH, the contents of other dietary minerals, and three-way interactions between phytate, proteolytic enzymes and protein-AA in the digestive contents.
In this study, the ileal as well as total-tract digestibilities were determined in the same animals. Huisman et al. (1985) found no effect of the insertion of ileocecal reentrant cannulas on the ATTD of nutrients in pigs. In addition, Drochner (1984) in studies with mini pigs found no effect of the placement of ileal reentrant cannulas on the ATTD of nutrients. Further, recent studies by W. C. Sauer (unpublished results) showed no differences in the ATTD of CP and energy between pigs fitted with postvalvular T-cecum cannulas and intact pigs fed a corn-barley-soybean meal-peas diet.
There were no improvements in the AID of CP and AA with phytase supplementation to the diets in Exp. 1, 2, and 4 (Tables 3, 4, and 6). However, there were significant improvements or tendencies for improvements in the AID of CP and AA, and the contents of DE in Exp. 3, in which phytase was supplemented to a wheat-canola meal-soybean meal diet (Table 5).
The effect of phytase supplementation on the AID of AA was also examined in other studies with growing pigs (Mroz et al., 1994; Kemme et al., 1999; Traylor et al., 2001). Phytase supplementation to a "wet barley protein with fiber" diet (250 and 500 FTU/kg) did not affect the AID of AA in the study by Valaja et al. (1998). It should be mentioned that the phytate content in the "wet barley protein with fiber" diet was very low. Rice (2002) also showed no effect of phytase supplementation to an 11% CP diet on the AID of AA. Further, Sands (2002) reported no differences in the AID of AA when phytase (1,200 FTU/kg) was supplemented to either a low (0.22%) or high (0.39%) phytate P diet. Moreover, Traylor et al. (2001) reported no increases in the AID of AA when phytase (500 to 1,500 FTU/kg) was supplemented to a semipurified soybean meal diet. Recently, Johnston et al. (2004) carried out studies in which phytase (500 FTU/kg) was supplemented to a corn-soybean meal diet that contained adequate or decreased levels of Ca and P. Compared with a diet adequate in Ca and P, there were significant improvements or tendencies (P = 0.01 to 0.10) toward improvements in the AID of the essential AA when phytase was supplemented to the diet with reduced levels of Ca and P. Phytase addition (main effect; to diets with adequate and decreased levels of Ca and P) tended (P = 0.09) to increase the AID of isoleucine and leucine.
Mroz et al. (1994) and Kemme et al. (1999) reported small increases in the AID of AA upon phytase supplementation. Of the essential AA, these increases were significant for arginine (2.5 pu) and methionine (3.9 pu) in the study by Mroz et al. (1994), and for isoleucine (2.1 pu), lysine (2.4 pu), threonine (2.9 pu), and tryptophan (4.4 pu) in the study by Kemme et al. (1999). In the study by Mroz et al. (1994), phytase was supplemented (800 FTU/kg) to a corn-tapioca-soybean meal-barley-peas diet. In the study by Kemme et al. (1999) phytase was supplemented (900 FTU/kg) to a corn-soybean meal diet. Kornegay et al. (1998) reported small linear increases in the AID of the essential AA when phytase, at rates of 250 FTU/kg and 500 FTU/kg, was supplemented to a corn-soybean meal diet. The linear increases were significant for valine, isoleucine, and arginine. Supplementation of phytase at a rate of 500 FTU/kg diet increased the AID of the essential AA from 1.9 to 4.0 pu. Conversely, Officer and Batterham (1992) reported relatively large increases in the AID of the indispensable plus semiessential AA when phytase was supplemented to a semipurified diet containing 40% Linola (UGG, Ltd., Winnipeg, Canada) meal for growing pigs. The AID of the AA increased by 4.0 to 13.0 pu; however, only the increases for lysine (12 pu) and histidine (12 pu) were significant.
The effect of phytase supplementation on the AID of lysine, methionine, and threonine reported in the aforementioned studies and in this study are summarized in Table 8. Lysine, methionine, and threonine were selected, as these are often first- or second-limiting in diets for pigs. In studies in which phytase was supplemented at more than one rate to the basal diet, the highest AID values are presented. With the exception of the study by Officer and Batterham (1992), the increases in the AID of lysine, methionine, and threonine were of a very small magnitude with phytase supplementation. They suggested that a substantial proportion of protein-AA in Linola meal might be bound by phytate bonds. In the study by Officer and Batterham (1992), it should be pointed out that the AID of lysine, methionine, and threonine in the basal diet were relatively low, ranging from 50 to 71. Expressed as percentage units, if improvements occur with phytase supplementation, these will be larger for diets in which the AID of AA are lower compared with diets in which the AID of AA are higher. It also should be kept in mind that the slaughter method was used to determine the AID of AA in the studies by Officer and Batterham (1992), whereas in the other studies, cannulation techniques were used. Not taking into account the results for lysine by Officer and Batterham (1992), there were usually small increases in the AID of lysine, methionine, and threonine with phytase supplementation. Significant differences were found in six instances.
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reported no improvements in the AID of AA when phytase was supplemented to either a high- or low-phytate P diet. These results were recently confirmed by Liao et al. (2004) in studies with growing pigs, in which phytase was also supplemented to either a high- or low-phytate P diet. The high-phytate diet contained 20% rice bran, which is rich in phytate P. Supplementation of phytase to either diet did not improve the AID of CP or AA. It is difficult to explain why there was a response to the diet used in Exp. 3 and not in the other experiments. The contents of phytate-P did not differ to a large extent among the diets. Taking into account the results by Sands (2002) and Liao et al. (2004), perhaps the content of phytate-P per se is not the primary determinant for a response to phytase. Rather, it may be the amount of protein (AA) that is complexed with phytate-P and which may differ among feed ingredients.
The effect of phytase supplementation on the ATTD of CP and content of DE was also examined. There were no significant increases in the ATTD of CP in Exp. 2 and 3. The content of DE tended to increase in Exp. 3. As was reviewed by Liao et al. (2002), phytate may decrease starch digestibility by several mechanisms. These include the formation of complexes with starch and inhibition of 
-amylase through direct binding. Supplementation of phytase may release starch and -amylase, thereby increasing the DE content of the diets.
In conclusion, this study showed that there was no consistent effect of phytase supplementation on the AID of AA in different diets for weanling pigs. Significant improvements or tendencies toward improvements were only observed in Exp. 3, when phytase was supplemented to a wheat-soybean meal-canola meal diet.
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Implications |
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TopAbstractIntroductionExperimental ProceduresResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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2 Financial support was provided by DSM Food Specialties, Delft, The Netherlands, the Alberta Livestock Industry Development Fund, Ltd., and the Alberta Agric. Res. Inst.
3 The authors thank K. Sauer and R. Engelbert for technical assistance.
4 Correspondence: 410, Agriculture and Forestry Center (phone: 780-492-7659; fax: 780-492-4265; e-mail: willem.sauer{at}ualberta.ca).
Received for publication June 25, 2004. Accepted for publication November 29, 2004.
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TopAbstractIntroductionExperimental ProceduresResultsDiscussionImplicationsLiterature Cited |
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