Effect of maize conservation technique and phytase supplementation on total tract apparent digestibility of phosphorus, calcium, ash, dry matter, organic matter and crude protein in growing pigs

Animal Feed Science and Technology 185 (2013) 70–77

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Effect of maize conservation technique and phytase supplementation on total tract apparent digestibility of phosphorus, calcium, ash, dry matter, organic matter and crude protein in growing pigs E. Humer, W. Wetscherek, C. Schwarz, K. Schedle ∗ Institute of Animal Nutrition, Products and Nutrition Physiology, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, A-1190 Vienna, Austria

a r t i c l e

i n f o

Article history: Received 25 October 2012 Received in revised form 2 July 2013 Accepted 7 July 2013

Keywords: Digestibility Fermentation Maize Phosphorus Phytase Pig

a b s t r a c t Two experiments were carried out to investigate the effect of different maize conservation techniques and phytase addition on coefficients of total tract apparent digestibility (CTTAD) of phosphorus (P), calcium (Ca), ash, dry matter (DM), organic matter (OM) and crude protein (CP). In the first experiment, 9 growing pigs were allotted to a triplicate 3 × 3 Latin square design to measure CTTAD of P in three different maize conserves. Maize was either dried (dried maize), ensiled after milling (maize silage) or tight-closed-stored as whole grain (TCS-maize). Diets consisted of the differently conserved maize alone supplemented with amino acids and a mineral and vitamin source. To achieve P-deficient diets, no P was added. In experiment 2 same experimental design, diet composition and feedstuffs were used as in the first study. However, in this study diets were supplemented with 750 FTU phytase kg−1 diet from Schizosaccharomyces pombe. To evaluate the effect of phytase supplementation, results of both experiments were pooled for statistical analysis. The obtained results show that the CTTAD of P was greater (P < 0.0001) when maize was fermented compared with dried maize (0.42 in maize silage, 0.37 in TCS-maize, 0.28 in dried maize). The CTTAD of P was enhanced in all conservation types through phytase addition (0.46 in dried maize, 0.55 in maize silage and 0.52 in TCS-maize) (P < 0.0001). Moreover, CTTAD of Ca was higher in the fermented maize groups (P < 0.0001) compared with dried maize. However, it was not influenced by phytase supplementation. The CTTAD of DM and OM were not influenced by fermentation. The CTTAD of CP was lower in fermented maize (P < 0.05) compared to dried maize, whereby this effect was more pronounced in maize silage. However, the CTTAD of DM, OM and CP were not influenced by phytase supplementation. In conclusion, maize fermentation in the form of ensiling increases CTTAD of P in growing pigs. Therefore lower P supplementations are required and P excretions with manure can be reduced. Although CTTAD of P is improved when phytase is added, fermentation still offers advantages compared to dried maize. © 2013 Elsevier B.V. All rights reserved.

Abbreviations: CTTAD, coefficient of total tract apparent digestibility; FTU, phytase activity expressed in units; TCS-maize, tight-closed-stored maize. ∗ Corresponding author. Tel.: +43 47654 6108; fax: +43 47654 6105. E-mail address: [email protected] (K. Schedle). 0377-8401/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anifeedsci.2013.07.001

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1. Introduction In commercial pig diets the major ingredients are grains, seeds or products from seeds. The majority of phosphorus (P) in these feedstuffs occurs in the form of phytates, the salts of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate). On the basis of the negative charge of this ester of phosphoric acid and inositol, phytic acid forms a complex salt called phytate with divalent metal cations such as calcium (Ca), iron, zinc, magnesium and manganese and therefore inhibits the absorption of these minerals (Kornegay, 2001). The enzyme phytase catalyzes the stepwise hydrolysis of phytate to inorganic phosphate and lower phosphorylated inositol phosphates (Lopez et al., 2000). Monogastric animals lack intestinal phytase enzymes at the level needed to adequately break down phytate-bound P. Due to the fact that only released o-phosphate (PO4 ) from the dephosphorylation reaction can pass through the gastrointestinal wall, the majority of plant-derived P is unavailable for pigs and will be excreted (Yi and Kornegay, 1996). Maize is one of the most important feed ingredients in swine diets. According to Nuss and Tanumihardjo (2010) phytic acid makes up 60–90% of P in maize and nearly 90% is located in the germ (Laboure et al., 1993). Maize contains negligible phytase activity, in the amount of 0–56 phytase units (FTU) kg−1 (Eeckhout and Depaepe, 1994; Weremko et al., 1997; Leytem and Thacker, 2010). Based on own calculations relating to analyzed phytate contents in feed ingredients by Pontoppidan et al. (2007) maize contributes to about 60% of total phytate content in commercial maize-soybean meal pig diets. It is well known that storage procedures using fermentation techniques can lead to phytate degradation in seeds, whereby P absorption in monogastric animals increases (Niven et al., 2007; Blaabjerg et al., 2007). Although almost no phytase is present in maize, fermentation might improve CTTAD of P, due to the fact that lactic acid bacteria are able to degrade phytate (Lopez et al., 2000). In Austrian’s and South German’s pig production maize storage in an ensiled form is well established. Nevertheless, the common method to improve P digestibility in plant derived feedstuffs for monogastric animals is the supplementation with exogenous phytase as reviewed by Selle and Ravindran (2008). Hence, the aim of the present studies was to determine the effect of maize drying, maize ensiling after milling and tight-closed storage of whole grain with or without phytase application briefly before feeding on CTTAD of P, Ca, ash, dry matter (DM), organic matter (OM) and crude protein (CP) in growing pigs. Hence, the objectives of the current experiments were, to test the following hypothesis: Fermentation of maize in the form of maize silage and tight-closed stored maize improves CTTAD of P, Ca, ash, DM, OM and CP. Phytase supplementation improves CTTAD of P, Ca, ash, DM, OM and CP beyond the level reached via fermentation. To our knowledge, we are the first who investigated the effect of this three maize conservation techniques on CTTAD of P, Ca, ash, DM, OM and CP in growing pigs. 2. Materials and methods 2.1. Animals and housing The protocol of the pig studies was approved by the Austrian ministry for science and research and by the University of Natural Resources and Life Sciences, Vienna (BMWF-66.016/0010-II/3b/2012). Two consecutive experiments were conducted following triplicate 3 × 3 Latin square designs. Each study was carried out at the SRC (Lichtenwörth, Austria) with 9 different crossbred barrows, progeny of (Duroc x Landrace) x Piétrain. Animals were allotted randomly according to body weight (BW) and litter (pigs from 3 different litters were used each time) to individual metabolic cages (Ehret, Tulln, Austria). The experiments started with a 7-day adaption period to the metabolism cages, where a commercial diet for growing pigs based on maize and soybean meal was fed ad libitum. The stainless steel cages were equipped with wire mesh screens and drain pans for separate collection of feces and urine. Cage size was adjusted to the body size of each individual animal during the feeding trial. The barrows were fed equal amounts twice daily, at 7 AM and 6 PM. Feed intake was limited to 2.5 times of the metabolizable energy (ME) requirements for maintenance (GfE, 2006), based on the average BW of the pigs at the start of each experimental period. Pigs had free access to water throughout the whole experiment. Feces were collected quantitatively and stored at −20 ◦ C to prevent microbial activity until the end of each period. Urine samples were collected twice daily. The total amount was weighed, aliquots were taken, immediately frozen and stored at −20 ◦ C. At the end of each experimental period samples were thawed, pooled within animal and diet, and a subsample was retained for further analysis of P and Ca. 2.2. Maize conservation and diet formulation Maize used (P9400, Pioneer, Parndorf, Austria) was dried (“dried maize”), milled with a conventional mill and ensiled (“maize silage”) or tight-closed-stored as whole grain (“TCS-maize”), respectively. Maize temperature did not exceed 55 ◦ C during the drying process. Maize silage was prepared by air-proofed storage of milled maize under standardized conditions (grinding, compressing, air-tight closing). In contrast TCS-maize was kept in air-tight 6 L wide-neck-kegs as whole-grain (Bär, Salzburg, Austria). Fermented maize was stored for 56 days before the experiments started. Diets were formulated according to the guidelines of GfE (1994) for determination of CTTAD of P. They consisted of the maize alone with addition of amino acids (AA) and a mineral and vitamin source. To avoid regulatory induced P excretion, no P was supplemented. Limestone was added, to achieve the recommended Ca content of 6 g kg−1 DM. Ingredient composition of the diets is shown in Table 1. Due to the differences in DM content among maize conserves, varying amounts of conserved maize were mixed

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Table 1 Composition of test diets (dry matter basis). Ingredient

Amount, g kg−1

Maize L-Lys-HCl DL-Met L-Thr L-Trp Limestone Salt Vitamin premixa Trace mineral premixb

963.40 10.45 1.75 3.72 1.70 14.70 2.40 0.90 0.98

a Provided the following quantities of vitamins per kilogram of diet on dry matter basis: 10,050 I.U. vitamin A, 2001 I.U. vitamin D3 , 3.31 mg vitamin B1 , 5.33 mg vitamin B2 , 3.68 mg vitamin B6 , 0.0222 mg Vitamin B12 , 3 mg vitamin K3 , 55.22 mg nicotinic acid, 0.5049 mg folic acid, 14.25 mg pantothenic acid, 0.0738 mg biotin, 500 mg cholin, 100 I.U. vitamin E. b Provided the following quantities of minerals per kilogram of complete diet on dry matter basis: 119.34 mg Zn, 24.60 mg Cu, 60.08 mg Mn, 0.99 mg Co, 1.23 mg J, 0.41 mg Se, 119.34 mg Fe.

with the premix, to achieve a constant maize-premix proportion on DM basis. Maize and TCS-maize were briefly grounded with a conventional mill before feeding. All maize conserves were grounded in order to achieve the recommended particle size of Kamphues (2002) (>20% of the particles greater than 1 mm, <35% smaller than 0.2 mm). 2.3. Experiment 1 Nine crossbred barrows with an initial BW of 30.81 ± 0.89 kg were used to determine the effect of different maize conservation techniques (dried maize, maize silage, TCS-maize) on CTTAD of P, Ca, ash, DM, OM and CP. Each experimental period comprised 14 days, 7 days of diet adaption and 7 days sampling period. 2.4. Experiment 2 Referring to experiment 1, 9 growing barrows (BW: 34.57 ± 0.59 kg) were used to determine the effect of phytase supplementation of dried maize, maize silage and TCS-maize on CTTAD of P, Ca, ash, DM, OM and CP. Each experimental period lasted 13 days with 7 days adaption and 6 days sampling period. In contrast to experiment 1, diets were formulated with supplementation of 0.15 g microbial phytase from Schizosaccharomyces pombe (Phyzyme® XP 5000 G, Danisco, Marlborough, England) per kg complete diet, to achieve a phytase activity of 750 FTU kg−1 DM. Phytase was mixed with the premix at the beginning of the experiment and added to the maize conserves briefly before feeding. 2.5. Analytical methods Feed samples were taken at the beginning of each experimental period. After each collecting period feces were defrosted, homogenized and a fresh sample was used for determination of DM and CP. Another subsample of feces as well as the urine samples were freeze dried for further analysis. Phosphorus was determined photometrically (U5100-SpectrophotometerHitatchi, Metrohm, Wien, Austria) using the vanado-molybdate method to measure color intensity at 436 nm and Ca was determined by flame atomic absorption spectrophotometry (AAnalyst 200, Perkin Elmer, Brunn am Gebirge, Austria) after wet-ashing of lyophilized samples via microwave (MLS-ETHOS plus Terminal 320, Leutkirch, Germany). Ash, DM, OM, CP in feces and nutrients as well as pH in feeds were analyzed according to the official methods of VDLUFA (Naumann and Bassler, 2012). All chemical analyses were performed in duplicate. Phytase activity of feeds was measured according to the ISO 30024 method and phytate P was determined according to the AOAC method 986.11 with some modifications (Latta and Eskin, 1980). To determine fermentation characteristics, organic acids were measured in maize silage and TCS-maize by gas chromatography (Varian GC 3900, München, Germany) according to Zhao et al. (2006). Ammonia-N concentration was determined via UV-test, using a commercially available kit (R-Biopharm, Darmstadt, Germany). Amino acid composition was analyzed by the methods of Altmann (1992). 2.6. Calculations and statistical analysis The CTTAD of P, Ca, ash, DM, OM and CP were measured directly and calculated using the following equation: CTTAD = [(Fi − Ff )/Fi ], CTTAD = coefficient of total tract apparent digestibility of P, Ca, Ash, DM, OM or CP; Fi = total intake of P (g), Ca (g), Ash (g), DM (g), OM (g) or CP (g) during the collecting period; Ff = total fecal output of P (g), Ca (g), Ash (g), DM (g), OM (g) or CP (g) originating from feed that was fed during the collecting period. Data were subjected to ANOVA using the MIXED procedure of SAS, according to the following model: xijkl =  + Ti + BW + Pj + Ak + El + eijkl ,

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xijkl = dependent variable,  = overall mean, Ti = effect of treatments (i = dried maize, maize silage, TCS-maize, dried maize + phytase, maize silage + phytase, TCS-maize + phytase), BW = effect of BW at the start of the relevant experimental period, Pj = random effect of experimental period (j = 1, 2, 3, 4, 5, 6), Ak = random effect of animals (k = 1, 2, . . ., 18), El = random effect of experiment (l = 1, 2), eijkl = residual experimental error. Treatment means were separated using the least-squares means statement and differences were determined using the Tukey–Kramer test. Furthermore, the significance of the overall effect of fermentation was tested for all variables (linear contrast involving the average of the two fermentation groups vs. control, whereby the effects of phytase supplementation, experimental period, individual animal and BW were included in the statistical model). Statistically significant differences (P < 0.05) among least-squares means were indicated by superscripts. Differences with P-values < 0.1 and >0.05 were considered as a tendency. 3. Results 3.1. Chemical analysis of test feedstuffs Average analyzed nutrient concentrations of the different maize conserves, concentrations of AA and fermentation characteristics of the ensiled grains are shown in Table 2. Maize silage and TCS-maize contained less sugar and CP than dried Table 2 Analyzed nutrient composition, concentration of amino acids and fermentation characteristics of different maize conserves stored for 56 days (dry matter basis). Dried maize

Maize silage

TCS-maize

Nutrients Dry matter (g kg−1 ) Crude protein (g kg−1 ) Ether extract (g kg−1 ) Crude fiber (g kg−1 ) Ash (g kg−1 ) Starch (g kg−1 ) Sugar (g kg−1 ) P (g kg−1 ) Phytate P (g kg−1 ) Ca (g kg−1 )

882.9 101.1 42.1 18.0 14.8 714.8 21.2 3.23 2.28 0.11

775.0 96.8 39.2 14.6 15.2 737.5 10.7 3.37 1.90 0.16

745.4 98.4 36.6 18.8 14.5 726.6 5.7 3.29 2.10 0.11

Amino acids (g kg−1 ) Indispensable AA Lys Met Thr Trp Val Ile Leu His Phe Arg Total

2.54 2.22 3.15 0.69 4.63 3.38 11.71 2.66 4.65 4.22 39.85

2.42 2.20 3.06 0.67 4.55 3.26 11.07 2.43 4.48 4.12 38.26

2.42 2.22 3.17 0.69 4.70 3.38 11.59 2.54 4.60 4.19 39.50

Dispensable AA Ala Asp Cys Glu Gly Pro Ser Tyr

7.09 6.11 2.40 18.43 3.50 n.d. 3.92 3.05

6.68 6.07 2.36 16.99 3.44 n.d. 3.73 2.88

7.31 6.13 2.33 17.61 3.54 n.d. 3.89 3.09

Total All AA

44.50 84.35

42.15 80.41

43.90 83.40

Organic acids Acetic acid (g kg−1 ) Butyric acid (g kg−1 ) Lactic acid (g kg−1 )

n.a. n.a. n.a.

12.8 0.9 24.7

3.3 n.d. 18.6

Further parameters NH3 (g kg−1 N) pH

29.7 5.5

39.2 4.1

55.6 4.2

Abbreviations: n.a., not analyzed; n.d., not detectable; TCS-maize, tight-closed-stored maize.

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Table 3 Coefficient of total tract apparent digestibility of P, Ca, ash, DM, OM and CP, P- and Ca-content in urine, dry matter of feces and zootechnical performance. Diet Maize conservation

Dried maize

Maize silage

TCS-maize

Phytase1 Replicates per treatment

− n=3

+ n=3

− n=3

+ n=3

− n=3

+ n=3

SEM

CTTAD of P CTTAD of Ca CTTAD of ash P in urine (g kg−1 DM) Ca in urine (g kg−1 DM) CTTAD of DM DM of feces (g kg−1 ) CTTAD of OM CTTAD of CP ADG, g d−1 F:G, g kg−1

0.28 0.61 0.56 0.19 66.53a 0.89 397.3 0.90 0.81 331.6 3.62

0.46 0.66 0.61 0.51 47.40c 0.89 348.9 0.90 0.80 355.1 3.30

0.42 0.71 0.63 0.24 59.76b 0.89 361.8 0.90 0.77 394.7 2.85

0.55 0.71 0.67 0.72 26.31e 0.89 345.5 0.90 0.79 404.0 3.17

0.37 0.68 0.58 0.19 66.89a 0.89 365.3 0.89 0.79 367.3 3.01

0.52 0.71 0.65 0.60 40.21d 0.88 328.2 0.89 0.80 390.4 3.11

0.0166 0.0121 0.0785 0.0348 2.2755 0.0019 4.4557 0.0018 0.0056 16.9249 0.1089

Statistical analyses P-value C

P-value Phy

P-value C × Phy

P-value F2

<0.0001 <0.0001 <0.0001 0.0048 <0.0001 0.0433 0.0004 0.0178 0.0084 0.1513 0.2098

<0.0001 0.4080 0.0236 0.0099 <0.0001 0.4214 0.0085 0.8546 0.7819 0.7516 0.9600

0.3311 0.2233 0.4026 0.1301 0.0008 0.4246 0.0557 0.5490 0.0711 0.9157 0.3204

<0.0001 <0.0001 <0.0001 0.0138 <0.0001 0.8991 0.0006 0.4748 0.0164 0.0086 0.0304

Abbreviations: C, Conservation technique; CTTAD, coefficient of total tract apparent digestibility; F, Fermentation; Phy, Phytase supplementation; TCS-maize, tight-closed-stored maize; −, no phytase supplemented; +, phytase supplemented. 1 750 FTU kg−1 diet from Schizosaccharomyces pombe. 2 Contrast fermented versus dried maize. a,b,c,d,e Different superscripts indicate significant differences between conservation techniques.

maize, but since only two samples per conserve were analyzed, no statistical analysis was performed. Fermentation led to a slight decrease in AA concentrations, whereby relative degradation compared to dried maize was highest for histidine, glutamine and alanine in maize silage and lysine, histidine and glutamine in TCS maize, respectively. Phytate content was lower in fermented maize compared to dried maize (−17% in maize silage, −8% in TCS maize). With a DM content of around 760 g kg−1 the pH-value in fermented maize was decreased from 5.5 to 4. More lactic acid was generated in maize silage compared to TCS maize. Phytase activity in the different maize conserves was below the detection limit of 80 FTU kg−1 . In the second experiment an activity of 897 FTU kg−1 on average (840 ± 200 FTU kg−1 in dried maize, 610 ± 200 FTU kg−1 in maize silage, 1240 ± 249 FTU kg−1 in TCS-maize) was analyzed. 3.2. Results of the digestibility trials All 18 animals stayed healthy throughout the experiments. Results of the CTTAD are given in Table 3. The CTTAD of P was lowest in dried maize without phytase supplementation (0.28). Maize ensiling after milling as well as tight-closed storage without phytase significantly increased CTTAD of P (+50% in maize silage and +32% in TCS-maize compared to dried maize) (P < 0.0001). Generally, CTTAD of P in maize conserves reached higher CTTAD values when phytase was supplemented (P < 0.0001). Phytase supplementation to dried maize resulted in a CTTAD of P of 0.46. Nevertheless, higher CTTAD values were recorded in maize silage (+20%) and TCS-maize (+13%). Moreover, CTTAD of Ca was lowest in animals fed diets with dried maize (0.61), compared to maize silage (+16%) and TCS-maize (+12%) (P < 0.0001). Phytase supplementation showed no additional beneficial effect (P > 0.10) on CTTAD of Ca. Fermentation as well as phytase supplementation improved CTTAD of ash (P < 0.05). A similar picture was shown for the parameter P-content in urine, which was higher in fermented maize (P < 0.05) and in phytase-supplemented maize, respectively (P < 0.01). On the opposite, urinary Ca content was lowered through fermentation as well as phytase supplementation (P < 0.0001). The observed interaction between conservation techniques and phytase supplementation (P < 0.001) for this investigated parameter seems interesting. Compared to the highest urinary Ca content, which was found in phytase unsupplemented dried maize and TCS-maize, the phytase supplemented maize silage group contained 61% less Ca in urine. Dry matter of feces was highest in the dried maize diet without phytase supplementation (P < 0.001). The CTTAD of DM and OM were lower in TCS-maize compared to maize silage (P < 0.05). The CTTAD of CP was lower in fermented maize compared to dried maize if no phytase was supplemented, whereby this effect was more pronounced in maize silage (P < 0.01). Phytase addition affected neither CTTAD of DM and OM nor CTTAD of CP. However, phytase supplementation and conservation techniques tended to interactively effect DM of feces and CTTAD of CP (P < 0.10). Average daily gain (ADG) and feed to gain ratio (F:G) were improved if maize was fermented (P < 0.05). No significant improvement in zootechnical performance was observed in phytase supplemented diets. 4. Discussion The present study was undertaken to describe the effect of maize conservation technique with and without phytase supplementation on CTTAD of P, Ca, ash, DM, OM and CP in growing pigs. During air-proofed storage moist maize undergoes mainly lactic acid fermentation (Taylor and Kung, 2002). Due to the fact that this process leads to phytate degradation in

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seeds P utilization in monogastrics might be improved (Niven et al., 2007). This may subsequently reduce feed costs for the farmer and P pollution. The obtained CTTAD of P (0.28) for dried maize is comparatively high for maize without phytase addition. Nevertheless, the value is in the order of magnitude for pigs fed maize-based diets, which range from 0.12 to 0.29, as observed by other authors (Almeida and Stein, 2010; Bohlke et al., 2005; Calvert et al., 1978). The differences in CTTAD of P in normal maize measured in several experiments indicate varying amounts of phytate P between maize varieties. Maize ensiling led to a significant enhancement in CTTAD of P and Ca. Our results are in accordance with Bohlke et al. (2005) and Spencer et al. (2000), who determined a higher CTTAD of P and Ca in low-phytate maize compared to normal maize. Thus the hypothesis that the CTTAD of P and Ca can be enhanced by fermentation is supported. It is noteworthy that the increase in CTTAD of P in maize silage and TCS-maize corresponded well with the extent of phytate degradation. One possible explanation for the increase in CTTAD of P and Ca in fermented maize diets could be the reason that lactic acid bacteria are able to decrease phytic acid contents (Lopez et al., 2000), due to their ability to produce phytase (De Angelis et al., 2010). Niven et al. (2007) found a 38% decrease in insoluble P in high moisture maize during storage. The DM content of 750 g kg−1 and detected content of 44% soluble P in their study is similar to our DM content of 760 g kg−1 and content of non-phytate P of 40% on average in fermented corn. Furthermore, Pieper et al. (2011) measured a higher CTTAD of P in pigs fed moist triticale and wheat supplemented with lactic acid bacteria compared to dry grains. Those results might indicate a decrease in phytic acid content during ensiling. However, it is noteworthy that triticale and wheat are rich in plant phytase, therefore phytate degradation might have taken place at moist conditions before ensiling. In their study a lactic acid concentration of about 20 g kg−1 in grains with approximately 750 g DM kg−1 led to the highest CTTAD, whereas a higher concentration of approximately 33 g kg−1 in high moisture grains (about 650 g DM kg−1 ) failed to increase the CTTAD compared to dried grains. The ability of natural lactic acid fermentation to reduce the phytate content is dependent on various conditions (type and metabolic activity of fermenting organisms, solids/water ratio, incubation temperature, etc.) and therefore difficult to determine. Moreover, these conditions will influence the activity of phytases of the host material (Chavan and Kadam, 1989; Hotz and Gibson, 2001). The insufficiency of the lactic acid concentration of 33 g kg−1 DM to increase CTTAD of P significantly compared to the dried grains in the study by Pieper et al. (2011) might be explained by the fact that the faster decline in pH compared to low moisture grains was less favorable for endogenous as well as lactic acid phytase activity (Carlson and Poulsen, 2003; Zamudio et al., 2001). Consequently, the lactic acid concentration of about 22 g kg−1 and DM contents of 760 g kg−1 on average in fermented maize in our study seem to be optimal for high CTTAD of P by the application of the fermentation technique. A further explanation for the hydrolysis of phytate bound P during fermentation might be the activation of endogenous phytase activity. Maize phytase activity is very low and is mainly influenced by temperature and pH (Konietzny and Greiner, 2002). Considering that several studies (Chang, 1967; Scheuermann et al., 1988; Laboure et al., 1993) indicate a slightly acidic pH (∼5) as optimum, it can be assumed that favorable conditions occurred in the fermentation process and hence naturally occurring phytase might have been activated (Kozlowska et al., 1996). In our study a pH value of approximately 4 for fermented maize was recorded. Nevertheless, irrespective of conservation type phytase activity in maize was not detectable. This finding is consistent with other experiments, where maize showed no phytase activity below our detection limit of 80 FTU kg−1 (Eeckhout and Depaepe, 1994; Leytem and Thacker, 2010; Weremko et al., 1997). Nevertheless, on the basis of our results, we can conclude that phytate degradation might be mostly influenced by the lactic acid bacteria and not through the activation of the maize phytase by a decreased pH value. Phytase addition led to an increase in CTTAD of P in maize. Microbial phytase improved CTTAD of P relatively more in the dried maize, as CTTAD of P was enhanced by 64% compared to 31% in maize silage and 41% in TCS-maize. The reason for this observation might be that less phytate was available for degradation in maize silage and TCS-maize, because some of the phytate in this conserves was degraded before exogenous phytase was supplemented. However, although the enhancement was not so pronounced in ensiled maize, still differences in respect of conservation method remained. The findings of the present study are supported by several other authors (Fandrejewski et al., 1997; Jendza et al., 2005; Mroz et al., 1994), who found an enhanced CTTAD of P in maize-based diets in pigs through phytase supplementation. Jendza et al. (2005) were also able to detect an improved CTTAD of Ca in diets supplemented with 500 and 1000 FTU kg−1 of Escherichia coli phytase and Mroz et al. (1994) with addition of 800 FTU kg−1 from Aspergillus niger phytase, respectively. This is in contrast to our study. The sufficient Ca supply might be a possible reason, why this effect was not as pronounced in our study. Therefore, the hypothesis that CTTAD of P will increase if maize conserves are supplemented with phytase is supported. Phosphorus supply under the requirement results in a decrease of P excretion via urine, because increased P reabsorption in the kidneys takes place (Columbus et al., 2010). At higher levels of digestible P, increased P content in urine was recorded. Similar results were observed by Columbus et al. (2010) and Stein et al. (2006). The overall low P contents in urine indicate that the concentration of P in the diets used in the current experiments did not exceed the pig’s requirement for maximum P retention (Stein et al., 2006). In contrast to P the urinary content of Ca decreased as the concentration of digestible dietary P increased. This observation may be explained by the fact, that both Ca and P are needed for bone tissue synthesis. At lower levels of digestible P intake, more Ca is digestible than needed for bone formation. The extra Ca has to be excreted via urine (Stein et al., 2006). The reduced Ca content in urine and the lack of an effect on CTTAD of Ca due to phytase supplementation indicate that CTTAD of Ca increased in fermented maize indirectly through the increase in CTTAD of P. To our knowledge, we are the first who investigated the effect of these three maize conservation techniques on CTTAD of P, Ca, ash, DM, OM and CP in growing pigs. CTTAD of DM and OM were not influenced by fermentation. However, CP was lower in fermented maize, whereby this effect was less pronounced if phytase was supplemented. Pedersen and Stein

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(2010) were able to detect a tendency for improvement in the CTTAD of DM in pigs fed a fermented liquid maize-soybean meal diet compared to dry feeding, which may be explained by an enhanced fiber solubility and thereby fiber disappearance through enhanced microbial activity during fermentation (Pedersen and Lindberg, 2003). Thus, the low fiber content in our study may explain the missing positive effect of fermentation on CTTAD of DM and OM. Supplementary microbial phytase influenced neither CTTAD of DM, OM nor CP, which is in agreement with Johnston et al. (2004) and Liao et al. (2005), who found no increase in ATTD of N when 500 FTU kg−1 and 2000 FTU kg−1 of Aspergillus niger phytase, respectively were added to maize-based diets. Furthermore, Spencer et al. (2000) found no difference in CTTAD of N between high and low-phytate diets. In contrast to our findings Mroz et al. (1994) obtained an increase in CTTAD of DM, OM and CP through addition of 800 FTU kg−1 of Aspergillus niger phytase to a maize-tapioca-soybean meal diet in pigs and Fandrejewski et al. (1997) a higher CTTAD of nutrients if maize-soybean meal diets or maize-rapeseed meal diets were supplemented with 1000 FTU kg−1 of Aspergillus niger phytase. This controversy may arise from possible differences in dietary nutrient source, various configurations of phytate–mineral–nutrient complexes and/or diverse in binding affinity of present inositol phosphates. Based on the fact, that only CTTAD of ash was improved by fermentation and phytase addition, the hypothesis that fermentation as well as phytase supplementation enhance CTTAD of ash, DM, OM and CP is not completely supported. However, to compare CTTAD values with other studies, it must be taken into account that pigs had to be fed restricted to 2.5 times maintenance energy level according to the guidelines of GfE (1994). As Chastanet et al. (2007) demonstrated that feed restriction significantly increases CTTAD of DM, CTTAD values generated in this experiment may be overestimated. It is well known that P deficient diets decline zootechnical performance in pigs (Eeckhout et al., 1995). In our study ADG and F:G were improved by the fermentation process, which might indicate a more pronounced P deficiency in the dried maize diet. Likewise, Spencer et al. (2000) found an improvement in ADG in pigs fed low-phytate maize compared to normal maize. Finally, fermentation of maize might be of interest, as it offers potential benefits on mineral CTTAD. Results of the current study indicate that air-proofed storage of wet maize releases phytate-bound P. However, this release can be further enhanced by the addition of exogenous phytase. 5. Conclusions Ensiling of maize increases CTTAD of P in growing pigs and therefore reduces the requirement for P supplementations in swine diets and hence minimizes the amount excreted by the animal. 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