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ANIMAL NUTRITION |
,2
ebek
* DSM Food Specialties, R&D—FTD, 2600 MA Delft;
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Animal Nutrition Group, Wageningen University & Research Center, 6700 AH Wageningen;
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Division Nutrition and Food; and
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Division Applied Research, Animal Sciences Group, Wageningen University & Research Center, 8200 AB, Lelystad, The Netherlands
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: digestibility • dose-response • minerals • phytase • weaner pig
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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; Bebot-Brigaud et al., 1999). Phytase hydrolyzes the orthophosphate groups from phytate. Phytate-bound nutrients are liberated as well. The result is not only a greater digestibility of P but also of protein (Kies et al., 2001) and minerals. Dietary microbial phytase was reported to increase digestibility of Ca, Mg, Mn, Zn, Cu, and Fe in pigs at a dose level of 500 to 1,500 phytase units (FTU)/kg of feed (Pallauf et al., 1992; Adeola, 1995; Jongbloed et al., 1995).
There is a dearth of information about the effect of phytase doses greater than 1,500 FTU/kg on mineral digestibility. Düngelhoef and Rodehutscord (1995) and Kornegay (2001) estimated that only a small additional effect on P digestibility in pigs would be obtained at dose levels exceeding 1,500 FTU/kg. The effect of high phytase doses on P and Ca digestibility was studied in broiler chicks (Shirley and Edwards, 2003; Augspurger and Baker, 2004) and in pigs (Harper et al., 1999). The authors concluded that phytase continued to improve performance, bone characteristics, and P and Ca digestibility up to a dose of 10,000 (Harper et al., 1999; Augspurger and Baker, 2004) or 12,000 FTU/kg (Shirley and Edwards, 2003).
The present experiment was performed to obtain more information about the effect of graded phytase doses up to a high level (100, 250, 500, 750, 1,500, and 15,000 FTU/kg of feed) on the digestibility of P, Ca, Mg, Na, K, and Cu in weaner pigs.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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The ethics committee of DLO-Institute for Animal Science and Health approved the experimental protocol. Two hundred and twenty-four crossbred [(Yorkshire x Dutch Landrace) x Yorkshire] female and castrated male weaner pigs were used from weaning (at about 28 d of age; mean initial BW of 7.8 kg). Each of the 8 treatment groups consisted of 4 replicates (pens) with 7 pigs each. The 7 pigs in each pen (4 females and 3 castrates, or 3 females and 4 castrates) were selected from 7 pairs of sows and were chosen randomly. No littermates were used in any one pen. Selection criteria of the weaner pigs were health status, weight, and sex. Pigs remained in the farrowing house during the 43-d experiment. Four farrowing houses were used, each one being considered a block and each block containing 8 pens. The 8 treatments were randomly assigned to pens in each of the 4 blocks. Pen size was 1.6 x 1.8 m. Temperature at weaning was kept at 25°C and was lowered by <1°C every week thereafter. Ventilation was thermostatically controlled.
Treatments, Diets, and Feeding
Eight treatments were the basal diet containing 0 FTU of added phytase (basal diet), the basal diet to which 100, 250, 500, 750, 1,500, or 15,000 FTU of phytase was added per kilogram of feed (as-fed basis) and the basal diet to which monocalcium phosphate was added (positive control). The basal diet contained nutrients at or above the levels recommended by the CVB (2002), except for Ca and P. Digestible P (dP) content of the basal diet was estimated at 1.25 g/kg; its composition and proximate analyses are presented in Table 1. As the positive control, the basal diet was supplemented with 1.5 g of dP/kg in the form of monocalcium phosphate monohydrate (MCP), with an assumed P digestibility of 83% (CVB, 2000). One FTU is defined as the phytase activity that liberates 1 micromole of orthophosphate per minute from 5.1 mM sodium phytate at 37°C and at pH 5.5 (Engelen et al., 1994). The phytase source was Natuphos Granulate (DSM Food Specialties, Delft, The Netherlands) and was added based on its analyzed activity.
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. The analyzed and calculated values agreed well. Weaner pigs had ad libitum access to feed and normal tap water. Contents of Ca, P, Mg, Na, and K in the water were approximately 64, 0.01, 4.5, 19.8, and 1.0 mg/L, respectively. The Cu level was negligible. The impact of these minerals was ignored in the digestibility calculations.
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Body weights and intake were recorded on d 0, 8, 29, and 43. Health status of the pigs was monitored twice daily throughout the experiment. Fresh fecal grab samples were collected in wk 4 and 5; care was taken to collect feces from each of the different weaner pigs. Sampling was on Tuesdays and Thursdays at 0800 to 0830, 1000 to 1030, 1300 to 1330, and 1500 to 1530. The samples were pooled per pen per week and frozen at –18°C pending analysis. Feed was sampled during its production.
All analyses were performed on freeze-dried feed and fecal samples. Feeds were analyzed for DM, ash, Ca, P, Mg, Na, K, Cu, Cr, and phytase activity. Phytate-P and nitrogen were analyzed in the basal diet only. Feces were analyzed for DM, ash, Ca, P, Mg, Na, K, Cu, and Cr. Dry matter, ash, and nitrogen (Kjeldahl) were assayed using AOAC procedures (1984). Mineral levels (except Cr) were determined using inductively coupled plasma, atomic emission spectrometry, according to NEN-ISO (1998). The method of Williams et al. (1962) was used to analyze chromium. Phytate-P was measured by the enzymatic method (Bos et al., 1993) and phytase activity by the method of Engelen et al. (1994). Digestibility coefficients of DM, ash, and the minerals under investigation were calculated using Cr as an indigestible marker according to standard procedures.
Statistical Analysis
Data were analyzed by ANOVA as a randomized complete block design, with pens as the experimental units, using SAS Version 6.12 (SAS Inst., Inc., Cary, NC). Because no treatment x week interaction was observed for the digestibility coefficients, results for the 2-wk collection period were averaged and analyzed as such. The effect of phytase addition was tested using orthogonal polynomial contrasts and differences between selected treatments as single-df contrasts, at a significance level of P < 0.05. Negative exponential dose-response equations were fitted using the NLIN-procedure of SAS for performance and mineral digestibilities for all treatments excluding the positive control.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Few health problems occurred in this experiment. A limited number of animals were treated for lameness. Eight weaner pigs died, mainly due to edema disease. Mortality was not related to treatment. Average final weight of the weaner pigs was 27.5 kg.
Performance of pigs fed the positive control diet was significantly better than for those fed the basal diet (P < 0.001; Table 3). These results indicate that the basal diet was clearly deficient in dP. Dietary phytase supplementation affected ADFI, ADG, and G:F in a dose-dependent manner. The calculated exponential relationships were:
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in which FTU is the added microbial phytase activity (FTU/kg).
Digestibility Values
Treatment affected the digestibility of all measured response criteria (P < 0.01; Table 4). Dry matter digestibility was slightly lower in the positive control diet than in the basal diet (P = 0.09) and the diet with 500 FTU/kg (P < 0.05), but differences between diets were small. Ash, P, Mg, Na, K, and Cu digestibility increased (P < 0.001) in a dose-dependent (linear and nonlinear) manner up to a level of 15,000 FTU/kg. Also, Ca digestibility increased in a dose-dependent manner, but this effect may be biased due to different limestone levels in the diets. Copper digestibility was negative at phytase doses below 750 FTU/kg. An exponential model was fitted to the dP levels, resulting in the following equation:
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The estimated dP level of the basal diet according to this model was 1.26 g/kg (measured was 1.22 g/kg; Table 2
). The plateau level was 3.02 g of dP/kg, which was reached at about 5,000 FTU/kg (3.04 g measured at 15,000 FTU/kg). Consequently, the maximal amount of dP generated by phytase in this diet was 1.76 g/kg.
Similar exponential models were fitted for the other minerals. Digestible DM gave no good fit, and digestible ash and Ca are biased due to the different inclusion levels of limestone. These equations are, therefore, not presented. The equations for Mg, Na, K, and Cu are as follows:
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The amounts of these minerals digested due to phytase can easily be calculated from these equations.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). The animals in this experiment will not show their maximal performance because dP contents of all diets were below their requirement (Jongbloed, 1987). Phytase increased P digestibility and improved performance, which is in agreement with previous studies (Beers and Jongbloed, 1992; Cromwell et al., 1993; Kornegay, 2001). In the current experiment, performance improved up to a phytase inclusion level of 15,000 FTU/kg. The maximal effect of phytase supplementation on performance has been estimated to be in the range of 500 to 1,500 FTU/kg in basal diets with similar dP contents to the one in the present experiment (Beers and Jongbloed, 1992; Gentile et al., 2003). In a review, Kornegay (2001) came to a similar conclusion. At a phytase dose of 1,500 FTU/kg, dP level was similar to that of the positive control diet (2.6 g/kg). At this phytase level, feed intake and ADG tended to be improve compared with the positive control diet by 6.9 (P = 0.076) and 6.6% (P = 0.074), respectively. These tendencies may indicate some effect of phytase on performance beyond the dP-related effect (Kies et al., 2001).
The exponential models used showed an excellent fit to the data. Comparing the curve for dP with results calculated from Kornegay (2001; equation 1,500) shows a similar dose-effect of phytase up to a level of about 750 FTU/kg. After that, the curve calculated from Kornegay’s equation plateaus rapidly. The level of the calculated plateau (after correction for difference in P digestibility at 0 FTU/kg and for a diet containing 3.63 g P/kg) is about 0.8 g of dP/kg feed lower than the plateau of the curve calculated from the current experiment.
Supplementation of microbial phytase to low-P diets improved P digestibility. This confirms previous results (Jongbloed et al., 1992; Adeola et al., 1995; Kornegay and Qian, 1996). Generation of digestible P by phytase was, however, much greater than expected, especially at the greatest dose (Table 2). The assumed dP generation with the addition of 15,000 FTU/kg was 1.1 g/kg, but the measured value was 1.83 g/kg. In earlier dose-response experiments, maximum dP generation was found at much lower phytase doses. Beers and Jongbloed (1992) found a maximum at around 1,000 FTU/kg, both for corn-soybean meal and for by-product-based diets. Also, the curves calculated from the experiments of Kornegay and Qian (1996) and Yi et al. (1996) showed no further increase in P digestibility at doses greater than 1,000 to 1,500 FTU/kg of feed. Based on a literature review, Düngelhoef and Rodehutscord (1995) calculated a dose-response curve and concluded that little improvement in P digestibility at doses greater than 750 FTU/kg could be expected. The additional effect of phytase on P digestibility at a dose >1,500 FTU/kg was unexpected. The inclusion level of 15,000 FTU/kg was chosen to improve the estimation of the plateau level of the exponential curve. Because this model seems the most appropriate to describe the effect of phytase, all data points were included in estimation of the equation.
For P digestibility, results similar to those in current experiment were obtained in earlier experiments investigating very high doses of microbial phytase. Harper et al. (1999) concluded that a dose of 10,000 FTU/kg continued to improve performance, bone mineralization, and mineral digestibility of grower pigs. Augspurger and Baker (2004) reported improvements of performance and bone characteristics in broilers with phytase inclusion up to 10,000 FTU/kg, although they observed some differences between phytase sources. Shirley and Edwards (2003) conducted a broiler trial with phytase doses up to 12,000 FTU/kg. They observed that performance, bone characteristics, and P-retention improved with increasing phytase dose. In their study, phytate-P disappearance increased up to 85 and 95% at supplementations of 6,000 and 12,000 FTU/kg, respectively. These findings are comparable to our results in weaner pigs. Assuming a digestibility of 80% for nonphytate P (Jongbloed, 1987), phytate-P digestibility was 85% at 15,000 FTU/kg, compared with 14% in the basal diet (Table 2).
The continuing improvement of digestibility up to very high phytase doses was unexpected, given the earlier assumptions that the maximum effect would be realized at a dose of 1,000 to 1,500 FTU/kg. A possible explanation may be the following: Due to a number of factors, such as the residence time of feed and phytase, pH value, grade of phytate accessibility, and grade of phytase degradation in the stomach, and the phytase and phytate characteristics, the main site of phytase activity is the stomach (Jongbloed et al., 1992; Yi and Kornegay, 1996). In the case of a very high phytase dose (e.g., 15,000 FTU/kg), soluble phytate is the limiting factor in the biochemical reaction (Kemme, 1998). Phytate may be degraded faster or to a greater extent at such a high dose than is the case with normal doses of about 500 FTU/kg, in which soluble phytate is available in excess. One could imagine that the faster degradation of phytate creates space for additional phytate molecules to dissolve but that these molecules would not dissolve when a phytase dose of 500 FTU/kg was applied. The extra dissolved phytate molecules will be degraded by phytase.
Another speculation regarding the large effect at the high phytase dose is that a large part of the active phytase escapes the stomach to the small intestine. Phytase from Aspergillus niger has an optimal pH of 5.5 but retains 35 to 80% of its activity at pH values of 6.0 to 6.5 (Engelen et al., 1994). These pH values are observed in the upper half of the small intestine at 4.5 h after feeding (Van der Meulen and Bakker, 1991), which means that phytase can still be active in the small intestine. When phytase is included at 500 FTU/kg of feed, the activity in the small intestine is of limited magnitude, but in the case of a very high dose, the resulting phytase activity in the gut may be high, with consequently a greater level of phytate hydrolysis. An argument against this hypothesis could be that at greater pH phytate can precipitate with cations, e.g., Ca or Mg, depending on the mineral:phytate ratio (Cheryan, 1980). The solubility of Ca-phytate decreases rapidly at a pH above approximately 6, but the Mg salt precipitates at a greater pH. This indicates that in the upper small intestine, phytate is probably still available in a soluble form and that it is available for hydrolysis by phytase. De Rham and Jost (1979) showed that phytate might be soluble at pH from 5.5 to 11. Thus the hypothesized explanation for the additional effect at the high phytase dose on mineral digestibility, compared with the practical standard (500 FTU/kg) seems feasible. It needs to be tested in experiments in which phytate degradation and phytase activity in the duodenum of weaner pigs fed diets with a high phytase activity are measured.
Calcium digestibility increased with increasing levels of phytase addition, which is in agreement with earlier reports. O’Quinn et al. (1997) found a linear increase in Ca digestibility with increasing phytase supplementation up to 1,000 FTU/kg. The effect is, however, not always significant (Murry et al., 1997). In the current experiment, a constant Ca:dP ratio was realized in the diets by adding limestone to diets that contained microbial phytase. The observed increase in Ca digestibility was, therefore, confounded with limestone level. Jongbloed et al. (1995) showed increased Ca digestibility with phytase addition to the diet of grower pigs, but the increase was smaller at a high level of dietary Ca than at a low level. Digestibility of Mg and Cu increased with phytase supplementation. This is in agreement with earlier observations in weaner pigs (Pallauf et al., 1992; Adeola, 1995; Kies et al., 2005), in grower pigs (Jongbloed et al., 1995), and in sows (Jongbloed et al., 2004). The increased Ca digestibility due to phytase was not accounted for when formulating the diets. This may be a confounding factor on the estimated digestibility of minerals, because greater Ca levels may reduce digestibility of cations (Jongbloed et al., 1995). This would mean digestibility of Mg and Cu might be underestimated. Because diets in the current experiment did not contain excessive levels of minerals, this effect is likely of limited magnitude.
The improved digestibility of Na and K with phytase supplementation is surprising. Phytase addition increased fecal digestibility of these monovalent cations up to 10%-units (P < 0.001). This confirms recent findings in sows (Jongbloed et al., 2004) and in weaner pigs (Kies et al., 2005). The effect of phytase on digestibility of monovalent cations was not reported before those experiments. Sodium and K-phytates are highly soluble: Na-phytate dissolves more than 96% over the pH-range of 0.3 to 11.2 (Scheuermann et al., 1988). Probably for this reason, inhibition of Na and K-absorption by phytate has not been studied in vivo.
To obtain the maximal effect of phytase on animal performance, the increased mineral digestibility, including digestibility of Na and K, needs to be considered in practical feed formulation. Dersjant-Li et al. (2002) showed that the anioncation difference affects energy use in pigs, and Kies et al. (2005) calculated that increased absorption of minerals, and their subsequent excretion with urine, might increase energy expenditure. Even though this effect of minerals on energy use is small, it may have practical relevance.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Corresponding author: arie.kies{at}dsm.com
Received for publication May 27, 2005. Accepted for publication December 13, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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This article has been cited by other articles:
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  T. L. Veum and M. R. Ellersieck Effect of low doses of Aspergillus niger phytase on growth performance, bone strength, and nutrient absorption and excretion by growing and finishing swine fed corn-soybean meal diets deficient in available phosphorus and calcium J Anim Sci, April 1, 2008; 86(4): 858 - 870. [Abstract] [Full Text] [PDF]
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E. K. D. Nyannor, P. Williams, M. R. Bedford, and O. Adeola Corn expressing an Escherichia coli-derived phytase gene: A proof-of-concept nutritional study in pigs J Anim Sci, August 1, 2007; 85(8): 1946 - 1952. [Abstract] [Full Text] [PDF]
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