Interactions between supplemented mineral phosphorus and phytase on phytate hydrolysis and inositol phosphates in the small intestine of broilers1 ,2

Interactions between supplemented mineral phosphorus and phytase on phytate hydrolysis and inositol phosphates in the small intestine of broilers1,2 Ellen Zeller,∗ Margit Schollenberger,∗ Maren Witzig,∗ Yauheni Shastak,∗ Imke K¨ uhn,† Ludwig ‡ ∗,3 E. Hoelzle, and Markus Rodehutscord ∗

Institut f¨ ur Tierern¨ ahrung, Universit¨ at Hohenheim, 70599 Stuttgart, Germany; † AB Vista Feed Ingredients, ‡ ur Umwelt- und Tierhygiene, Universit¨ at Hohenheim, 70599 64293 Darmstadt, Germany; and Institut f¨ Stuttgart, Germany

Key words: broiler, phytate, phytase, inositol phosphates, mineral P 2015 Poultry Science :1–12 http://dx.doi.org/10.3382/ps/pev087

INTRODUCTION

tion depends on the breakdown of InsP6 . Phytases catalyze the hydrolysis of InsP6 to less-phosphorylated inositol phosphates (InsP5 , InsP4 , InsP3 , InsP2 , InsP1 ), myo-inositol, and orthophosphates; the hydrolysis of InsP1 to 5 isomers may also be catalyzed by other phosphatases. Phytases of different origins participate in different degradation pathways via various positional inositol phosphate isomers (InsPs). InsPs formed by different phytases have been assumed to possess physiological relevance in the digestive tract of broilers (Zyla et al., 2004). In the digestive tract of avian species, InsP degradation can be caused by the activity of dietary, endogenous mucosal, or microbiota-associated phytases and phosphatases. Recent studies have revealed the high capacity of broilers and their gut microbiota to hydrolyze InsP6 (Tamim and Angel, 2003; Leytem et al., 2008; Shastak et al., 2014; Zeller et al., 2015). However, endogenous and microbiota-associated phytase activity

In plant seeds and feedstuffs obtained from them, phytic acid [myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate); InsP6 ] is naturally present as phytate (any salt of InsP6 ), the primary storage form of organic phosphorus (P). Availability of this P frac C 2015 Poultry Science Association Inc. Received November 4, 2014. Accepted January 8, 2015. 1 This study has been funded in part by the Ministerium f¨ ur Wissenschaft, Forschung, und Kunst Baden-W¨ urttemberg, Stuttgart, Germany. The authors are grateful for the good suggestions made by Korinna Huber on the draft version of this manuscript. 2 Presented in part at the annual meeting of the Society of Nutrition Physiology, G¨ ottingen, Germany, March 18 to 20, 2014. Zeller, E., Schollenberger, M., K¨ uhn, I., and Rodehutscord, M. (2014): Interactions between supplements of monocalcium phosphate and a phytase on the presence of inositol phosphates in the ileum of broilers. Proc. Soc. Nutr. Physiol. 23:27. 3 Corresponding author: [email protected]

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between MCP and phytase supplements on myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6 ) hydrolysis (duodenum/jejunum: P ≤ 0.001; ileum: P = 0.004) and level of specific lower InsPs were detected. Supplementation with 12,500 FTU/kg phytase resulted in 92% InsP6 hydrolysis and strong degradation of InsP5 . This treatment resulted in higher P net absorption, affirmed by higher BW gain, tibia strength, and mineralization compared to treatments without or with 500 FTU/kg phytase (P ≤ 0.05). MCP supplementation reduced the degradation of InsP6 and specific lower InsPs in birds fed diets without or with 500 FTU/kg of phytase (P ≤ 0.05), but did not reduce InsP6 hydrolysis or degradation of InsP5 at the high phytase dose. Effects of added MCP on phytase efficacy depend on the dose of supplemented phytase. Differences in the concentrations of lower InsPs indicated that the initial step of InsP6 hydrolysis is not the only catabolic step that is influenced by MCP or phytase levels.

ABSTRACT Phytate breakdown in the digestive tract of broilers is affected by supplements of mineral phosphorus (P) and phytase with unknown interactions between the 2 factors. It was the objective to study phytate hydrolysis and the presence of inositol phosphate isomers (InsPs) as affected by supplements of mineral P and phytase in the small intestine of broilers. Fifteenday old broilers were assigned to 48 pens of 20 broilers each (n = 8 pens/treatment). Two low-P corn– soybean meal-based diets without (BD−; 4.4 g P/kg dry matter) or with monocalcium phosphate (MCP; BD+; 5.2 g P/kg dry matter) were supplied without or with added phytase at 500 or 12,500 FTU/kg. On d 24, digesta from the duodenum/jejunum and lower ileum was pooled per segment on a by-pen basis, freezedried, and analyzed for P, InsPs, and the marker TiO2 . Another 180 broilers (n = 6 pens/treatment, 10 birds each) were fed the 3 BD+ diets from d 1 to 21 to assess the influence of supplemented phytase on tibia mineralization and strength. Significant interactions

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ZELLER ET AL.

MATERIALS AND METHODS Study Diets Part 1 of the investigation was carried out with a 2 × 3 factorial arrangement of treatments [2 monocalcium phosphate (MCP) levels and 3 phytase levels]. Two basal diets were formulated to contain adequate levels of all nutrients according to the recommendations of the Gesellschaft f¨ ur Ern¨ ahrungsphysiologie (1999), with the exceptions of Ca and P. Corn and solvent-extracted

Table 1. Ingredient composition and analyzed nutrient concentrations of the basal diets. BD− Ingredients Corn Solvent-extracted soybean meal (48% CP) Soybean oil D,L-Methionine Monocalcium phosphate Sand Limestone Sodium chloride Choline chloride Sodium bicarbonate Vitamin mix1 Mineral mix2 Titanium dioxide Analyzed nutrient concentrations Dry matter Crude ash CP Crude fiber Ether extract ME (calculated) (MJ/kg)

BD+

g/kg as fed 594.5 340 30 2 5.5 14 1 2 3 2 1 5

594.5 340 30 2 3.5 16 1 2 3 2 1 5

g/kg as fed 898 55 206 19 68 12.6

897 54 209 21 66 12.6

1 Vitamin mix (Raiffeisen Kraftfutterwerke S¨ ud GmbH, W¨ urzburg, Germany), provided per kg of complete diet: 3.6 mg retinol; 75 μ g cholecalciferol; 30 mg α -tocopherol; 2.4 mg menadione; 3 mg thiamin; 6 mg riboflavin; 6 mg pyridoxine; 0.03 mg cyanocobalamin; 50 mg nicotinic acid; 14 mg pantothenic acid; 0.1 mg biotin; 1 mg pteroyl(mono)glutamic acid. 2 P, free mineral mix (Gelamin SG 1 Gefl¨ ugel, GFT MBH, Memmingen, Germany), provided per kg of complete diet: 15 mg Cu; 1.6 mg I; 90 mg Fe; 120 mg Mn; 80 mg Zn; 0.5 mg Se; 0.6 mg Co.

soybean meal were chosen as the main ingredients to obtain low concentrations of total P (tP) and low intrinsic phytase activity (Table 1). The basal diets had the same composition, with the exceptions of MCP (BD+ contained MCP; BD− contained no MCP), sand, and limestone. The concentrations of tP and Ca were calculated to be 4.5 and 6.5 g/kg, respectively, of DM in BD− and 5.4 and 7.9 g/kg DM, respectively, BD+. These levels were confirmed by chemical analysis (Table 2). The ratio of Ca to tP in BD+ and BD− was calculated to be 1.5:1, in accordance with the protocol for determining available P from the World’s Poultry Science Association (2013). P and Ca levels were adjusted by replacing sand with the required levels of limestone and MCP. Titanium dioxide was included at a rate of 5 g/kg as the indigestible marker, and the intended Ti concentration was confirmed by analysis. Diets were mixed in the certified feed mill facilities of Hohenheim University’s Agricultural Experiment Station, location Lindenh¨ ofe in Eningen, Germany. The basal diets were mixed in 2 separate lots, and each lot was divided into 3 equal parts. One part of each lot remained without phytase. The other parts were supplemented with a phytase-containing product at an intended activity of 500 or 12,500 FTU/kg of diet. The supplemented phytase product was a thermotolerant, modified Escherichia coli (E. coli) 6-phytase produced in Trichoderma reesei (Quantum Blue, EC 3.1.3.26, supplied by AB Vista, Marlborough, UK).

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and efficacy depend on various dietary factors (Angel et al., 2002; Applegate et al., 2003; Abudabos, 2012). In poultry, mineral P and Ca reduced InsP6 hydrolysis and P net absorption (Ballam et al., 1984; Mohammed et al., 1991; Tamim and Angel, 2003). We previously described the influence of mineral P and Ca on the InsP pattern in the intestine (Shastak et al., 2014). Diets used in the poultry industry are often supplemented with phytase at 500 to 750 FTU/kg feed. However, a substantial part of dietary InsP6 remains undegraded in the digestive tract, perhaps partly due to mineral P that is also present in the diet. Recently, high doses of phytase beyond the current industry standards (>2,500 FTU/kg) have attracted research attention in an attempt to maximize the hydrolysis of feed InsP6 (Cowieson et al., 2011). While these high doses of supplemented phytase resulted in >90% InsP6 hydrolysis in broilers (Shirley and Edwards, 2003), the effects of such high supplementation on lower InsPs remain to be elucidated. Although phytase may show different degradation kinetics for the various InsPs, likewise a strong and fast release of inorganic phosphate (Pi ) from lower InsPs may occur. In addition to this InsP-derived Pi , Pi from mineral sources usually is also present in the diet and interacts with phytase and other phosphatases via product inhibition. In theory, the inhibitory effect of supplemental P should depend on the amount of enzyme present, and thus the decrease in phytase efficacy due to mineral P may be negligible at very high phytase dosage. However, we are not aware of any study that has investigated the in vivo interactions between mineral P and different levels of phytase and their effects on InsP degradation, especially not at very high phytase dosages. Therefore, the objectives of this study were to investigate the effects of supplemented phytase, the effects of mineral P supplementation, and their interaction on InsP6 hydrolysis and the appearance of InsPs in the duodenum/jejunum and lower ileum of broilers (Part 1). The effect of supplemental phytase on tibia mineralization was additionally assessed (Part 2). Supplementation at levels several-fold higher than current phytase standard levels was hypothesized to result in very high InsP degradation in the small intestine. Inclusion of mineral P was expected to reduce InsP degradation less when the enzyme was supplemented at the high levels compared to standard levels.

PHYTATE DEGRADATION WITH PHOSPHATE AND PHYTASE

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Table 2. Analyzed characteristics of the study diets. BD− Phytase

0 1

Phytase activity (FTU/kg) Ca (g/kg DM) tP (g/kg DM) InsP6 -P (g/kg DM) Ins(1,2,3,4,6)P5 (μ mol/g DM) Ins(1,2,3,4,5)P5 (μ mol/g DM) Ins(1,2,4,5,6)P5 (μ mol/g DM) Ins(1,3,4,5,6)P5 (μ mol/g DM) InsP6 (μ mol/g DM) 1 2

500

< 100 6.0 4.4 2.7 0.2 0.5 1.1 0.1 14.7

2

620 6.5 4.4 2.8 0.2 0.6 1.1 0.1 15.1

BD+ 12,500

0

12,800 6.3 4.3 2.8 0.2 0.5 1.1 0.1 15.0

< 100 7.6 5.2 2.8 0.2 0.5 1.1 0.2 15.1

2

500

12,500

716 7.4 5.2 2.7 0.2 0.5 1.0 0.1 14.6

12,500 7.8 5.3 2.9 0.2 0.5 1.1 0.2 15.4

Determined at pH 4.5 and 60◦ C. Determined at pH 5.0 and 45◦ C according to Greiner and Egli (2003) (U/kg).

10 birds each, and raised on wood shavings (1.5 × 1.5 m; 6 pens/treatment). Those animals were fed the 3 BD+ diets from d 1 to 21. The BD− diets were not used in this part of the study in order to avoid severe P deficiency in the young birds. The room temperature was initially set at 34◦ C and stepwise reduced to 32◦ C on d 4, 30◦ C on d 8, 28◦ C on d 14, and 26◦ C on d 21. Artificial lighting was provided with an intensity of 10 lx. On d 1 and 2, the lighting was constant; from d 3 onward, 18 h of light were provided per day. Diets and tap water were offered for ad libitum consumption.

Sampling and Analytical Methods Animals and Management This study was separated into 2 parts to measure InsP degradation (Part 1) and the characteristics of tibia mineralization (Part 2). The study was performed in the Agricultural Experiment Station of Hohenheim University, location Lindenh¨ ofe in Eningen (Germany) and was approved by the Animal Welfare Commissioner of the University to be in accordance with the German welfare regulations. Birds underwent routine vaccination against coccidiosis, Newcastle disease, and infectious bursal disease on d 3, 10, and 14, respectively. In total, 1,140 unsexed broiler hatchlings (Ross 308) were obtained from a local hatchery (Br¨ uterei S¨ ud GmbH and Company KG, Regenstauf, Germany). Out of these, 960 hatchlings were randomly allocated to 48 pens (1.2 × 1.2 m), with 20 birds/pen (Part 1). Animals received a commercial starter diet until 15 d of age; this diet contained 0.90% Ca, 0.65% tP, 22.0% CP, 6.8% ether extract, 12.6 MJ ME/kg, and 606 FTU/kg 3phytase (EC 3.1.3.8, 4a E1600). On d 15, the birds were weighed and the 6 dietary treatments were assigned to the pens (8 pens/treatment) in accordance with a completely randomized block design in the animal house. Diets were fed until slaughter at d 24. For tibia measurements (Part 2), the remaining 180 hatchlings were randomly allocated to 18 floor pens,

For digesta collection at d 24 (Part 1), 15 out of the 20 birds from each pen were randomly selected, weighed, stunned with a gas mixture of 35% CO2 , 35% N2 , and 30% O2 , and euthanized via CO2 asphyxiation. To standardize feed intake prior to sampling, birds were deprived of feed 2 h before slaughter. One h before the birds were killed, the feed troughs were moved back into the pens on a pen-by-pen basis to ensure the same schedule for all pens. Samples from the duodenum and jejunum (pooled) and the lower part of the ileum [defined as the posterior two-thirds of the section between Meckel’s diverticulum and 2 cm anterior to the ileo-ceco-colonic junction (Rodehutscord et al., 2012)] were taken. The abdominal cavity was immediately opened and the entire digestive tract was removed, except the crop, and dissected. The digesta of the duodenum/jejunum and lower ileum was gently flushed out using double-distilled water (4◦ C). Samples were pooled for all birds of one pen separately for each segment, immediately frozen at −18◦ C, freeze-dried (Type Delta 1–24, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany), and pulverized as described for the feed. The pulverized samples were stored at 4◦ C until analysis. On d 21 of Part 2, the birds were stunned, and killed as described. The birds were weighed and the 3 closest to the average BW of the birds in each pen were chosen. The right tibiotarsus (tibia) of each bird was removed,

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Phytase was added as a premix containing the phytase product and a small amount of the respective basal diet. After additional mixing, the diets were pelleted through a 3 mm die without using steam. Pellet temperatures measured immediately after release from the press ranged between 57 and 69◦ C. Representative samples of the diets were taken for later analyses. The samples were pulverized using a vibrating cup mill (Type 6TOPF, Siebtechnik GmbH, M¨ ulheim–Ruhr, Germany) and stored at 4◦ C until analysis. The study diets contained similar concentrations of InsP5 and InsP6 (Table 2). Other InsPs were not detected in the diets. Phytase activity in BD− and BD+ was below the limit of detection (Table 2).

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ZELLER ET AL.

differences in detector response for InsP3 to 5 were used according to Skoglund et al. (1997). For the analysis of Pi concentrations in digesta 0.2 g sample was extracted with 10 mL of a solution containing 0.2 M NaF (pH 8.0) at 5◦ C for 15 min. The samples were centrifuged at 16,300 × g for 5 min at 4◦ C. For sample clean-up, the supernatant was filtered through a 0.2 μm cellulose acetate filter (VWR, Darmstadt, Germany) into a Microcon filter (cut-off 30 kDa; Millipore, Bedford, MA) and centrifuged at 16,300 × g for 30 min at 4◦ C. Filtrates were analyzed by high-performance ion chromatography and conductivity detection using a LC 3000 system (Dionex, Idstein, Germany) equipped with an Ion Pac 20 column and corresponding guard column. Activity of intrinsic and supplemented phytase in the diets was determined under enzyme-specific conditions. Intrinsic phytase activity in both basal diets was assayed according to the direct incubation method (quantification of liberated Pi ) according to Greiner and Egli (2003). In brief, samples were incubated in sodium acetate buffer containing 100 μmol sodium phytate at pH 5.0 and 45◦ C. Pi liberated within an incubation period of 20 to 40 min was measured spectrometrically using ammonium molybdate. In the phytase productcontaining diets, determination of phytase activity was assayed according to the validated method of the supplier (feed extracts analyzed at pH 4.5 and 60◦ C and units converted to FTU by a conversion factor of 5.67). The latter assay was run by Enzyme Services and Consultancy (Ystrad Mynach, Hengoed, UK).

Calculations and Statistical Analysis BW gain, ADFI, and feed-to-gain ratio were determined for each pen for d 1 to 21 (Part 2) or d 15 and 24 (Part 1). Feed-to-gain ratio was calculated with adjustment for mortality, which was recorded daily. InsP6 hydrolysis and P net absorption (y) were calculated on a by-pen basis according to the following equation: 

y(%) = 100 − 100 × 

Ti in the diet(g/kg DM) Ti in the digesta (g/kg DM)

InsP6 or tP in the digesta (g/kg DM) × InsP6 or tP in the diet (g/kg DM)





The amount of net absorbed P (g/kg DM) was calculated by multiplication of the tP concentration of the diet (g/kg DM) and the respective P (%) net absorption  divided by 100. The percentage of InsP , InsP , or 3 4   InsP5 in InsP3 to 5 was calculated as an indicator of the extent to which intermediary products with different degrees of phosphorylation (InsP3 to 5 ) were formed. Statistical analysis was performed using the MIXED procedure of the software package SAS for Windows (Version 9.1.3, SAS Institute, Cary, NC). Prior to statistical analysis, normality was assessed using the proc univariate procedure of SAS and homogeneity of the

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packed in a polyethylene bag, and frozen at −20◦ C. After defrosting, the adhering soft tissues, cartilage caps, and fibulae were manually removed and the bones were set aside. Tibiae were analyzed for cortical content via quantitative computed tomography using the single-energy peripheral Stratec XCT 960 bone scanner (Stratec Medizintechnik GmbH, Pforzheim, Germany) and for ultimate breaking strength using an Instron testing machine (Model 5565 with Bluehill Software, Instron Deutschland GmbH, Pfungstadt, Germany) and ash according to Shastak et al. (2012) with the following modifications: frozen tibiae were thawed at room temperature for approximately 90 min and subsequently used for measurement of cortical content and ultimate breaking strength. For the determination of ultimate breaking strength, the tibia was held by 2 supporters (spaced 28 mm apart), and physical power was applied to the midpoint of the bone by a static load cell (1 kN) with a crosshead speed of 200 mm/min. Broken tibiae were used for tibia ash determination. The concentrations of proximate nutrients in the feed were determined according to official methods in Germany (Verband Deutscher Landwirtschaftlicher Untersuchungs und Forschungsanstalten (2006)). Feed samples were analyzed for DM and crude ash (Method 3.1), CP (Method 4.1.1), ether extract (Method 5.1.1), and crude fiber (Method 6.1.1). The concentrations of Ca, tP, and Ti in diet and digesta samples were determined according to a modification of the sulfuric and nitric acid wet digestion procedure of Boguhn et al. (2009), as previously described (Zeller et al., 2015). Concentrations of InsPs in the diet and digesta samples were determined according to Zeller et al. (2015), with 2 modifications. Briefly, samples were extracted with a solution containing 0.2 M EDTA, 0.1 M NaF (pH 10), and 0.05 M Na2 HPO4 . For sample clean-up, the extracts were filtered through a 0.2 μm cellulose acetate filter (VWR, Darmstadt, Germany) into a Microcon filter (cut-off 30 kDa; Millipore, Bedford, MA) and centrifuged at 14,000× g for 30 min. Filtrates were analyzed with high-performance ion chromatography and UV detection at 290 nm after postcolumn derivatization using an ICS-3000 system (Dionex, Idstein, Germany). InsPs with different degrees of phosphorylation (InsP3 to 6 ) and their positional isomers were separated, without enantiomer differentiation, on a Carbo Pac PA 200 column and corresponding guard column. Fe(NO3 )3 solution in HClO4 was used as the reagent for derivatization, in accordance with Phillippy and Bland (1988). The elution order of InsPs was established using commercial standards if available. Clear identification of all InsP3 isomers was not possible. One peak was identified as Ins(1,5,6)P3 (out of the available standards). Another peak (InsP3 -a) corresponded in retention time to the retention time of Ins(1,2,6)P3 , Ins(1,4,5)P3 , and Ins(2,4,5)P3 (out of the available standards), all of which coeluted under the conditions used. InsP6 was used for quantification, and correction factors for

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PHYTATE DEGRADATION WITH PHOSPHATE AND PHYTASE Table 3. BW gain, feed consumption (FC) and feed-to-gain (F:G) ratio of broiler chickens between d 15 and 24 age.1 Phytase 0 2

BW gain , g/d FC, g/d F:G, g/g

500 c

72 107b 1.49a

b

77 112a 1.45a

MCP −

+

76 110 1.46

78 111 1.42

12,500 a

82 113a 1.39b

Pooled SEM

1.4 1.8 0.017

P-value Phy

MCP

Phy × MCP

< 0.001 < 0.001 < 0.001

0.028 0.79 0.026

0.49 0.86 0.52

1 Data are given as means (calculated across basal diets and phytase levels due to missing interactions) and pooled SEM. Each treatment had 8 pens with 15 (BW gain) or 20 birds (FC and F:G) per pen. 2 Average BW on d 15 was similar for all treatments (501 g). a–c Means in a row not sharing a common superscript differ significantly, P ≤ 0.05.

Table 4. InsP6 hydrolysis, P net absorption, and net absorbed P in different segments of the digestive tract of broiler chickens.1 BD+

0

500

12,500

Duodenum/jejunum Lower ileum P net absorption

55c 67c

67b 78b

91a 92a

Duodenum/jejunum Lower ileum2 Net absorbed P

20d 52

31b 58

52a 71

0.9 2.3d

1.4 2.5c

2.2 3.1b

InsP6 hydrolysis

Duodenum/jejunum2 Lower ileum

Pooled SEM

P-value Phy

MCP

Phy × MCP

1.6 3.2

< 0.001 < 0.001

0.003 < 0.001

< 0.001 0.004

50a 70

1.5 1.9

< 0.001 < 0.001

0.42 0.008

0.023 0.22

2.6 3.7a

0.07 0.09

< 0.001 < 0.001

< 0.001 < 0.001

0.18 0.007

0

500

12,500

48 d 51d

50d 58c,d

91a 92a

26c 47

31b 50

1.3 2.5cd

1.6 2.6c

%

%

g/kg DM

1 Data are given as means and pooled SEM, n = 8 pens per treatment with 15 birds per pen. BD− = Basal diet without monocalcium phosphate; BD+ = Basal diet containing monocalcium phosphate. 2 Means were significantly different between the 3 phytase levels (calculated across basal diets due to missing interactions), P ≤ 0.05. a–d Means in a row not sharing a common superscript differ significantly (multiple t-tests in case of interaction), P ≤ 0.05.

Table 5. BW gain, ADFI, and feed-to-gain ratio of broiler chickens between d 1 and 21 age and tibia variables at d 21.1 BD+ Phytase BW gain, g/d ADFI, g Feed per BW gain, g/g CC2 , mg/mm BBS3 , N Tibia dry weight, g Tibia ash weight, g Tibia ash, %

Pooled SEM

0

500

12,500

23c 33b 1.41 5.6c 79c 1.09c 0.44c 40b

31b 42a 1.35 8.5b 146b 1.42b 0.65b 46a

33a 44a 1.33 10.1a 176a 1.62a 0.75a 47a

P-value 0.4 1.0 0.024 0.27 6.4 0.034 0.018 0.5

< 0.001 < 0.001 0.09 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001

1 Data are given as means and pooled SEM, n = 6 pens per treatment with 10 birds per pen. BD+ = Basal diet containing monocalcium phosphate. 2 CC = Bone cortical content. 3 BBS = Bone breaking strength. a–c Means in a row not sharing a common superscript differ significantly according to Tukey’s test, P ≤ 0.05.

variance using residual plots. Data that showed nonnormally distributed residuals or heterogeneity of variance were log- or square-root transformed. Data expressed as percentages were arc-sine transformed. In Tables 3 to 7, nontransformed data were expressed as treatment means and their pooled SEM or SE. According to the 2 × 3 factorial arrangement of treatments (Part 1), the following statistical model was chosen: yijk = μ + αi + β j + (αβ )ij + rk + eijk , where yijk = kth observation of the ith level of MCP and jth

level of phytase, μ = general mean, αi = effect of the ith level of MCP (fixed), β j = effect of the jth level of phytase (fixed), (αβ )ij = interaction effect of MCP, level i, and phytase supplementation, level j, rk = effect of the kth block (random), and eijk = error term. Statistical significance was evaluated by two-way ANOVA (P ≤ 0.05). If an effect of interaction was detected (P ≤ 0.05), then differences between treatments were tested using multiple t-tests at a level of significance of P ≤ 0.05. For Part 2, the following statistical model was

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BD− Phytase

6

ZELLER ET AL. Table 6. Concentrations of different InsPs and Pi in the digesta of the duodenum/jejunum.1 BD− Phytase

Pooled SEM

500

12,500

0

500

12,500

nmol/g DM n.d. n.d.

n.d. n.d.

n.d. < LOQ

n.d. n.d.

115 < LOQ 0.03

184 n.d. 0.21

P-value Phy × MCP

Phy

MCP

0.17

.

.

0.022

< 0.001

.

.

110 0.034

0.042 0.06

0.006 0.92

0.13 0.71

0.001 < 0.001 < 0.001

0.025 0.48 < 0.001

0.88 0.27 0.034 0.10

32

n.d. n.d.

n.d. 361 0.19

n.d. 289 0.51

n.d. n.d.

n.d. 888 0.23

n.d. 461 0.53

162 580 321c n.d. 1.00

64 1,003 399b,c n.d. 0.81

n.d. 147 105d n.d. 0.49

213 487 512b n.d. 1.00

119 1,594 717a n.d. 0.74

n.d. 117 125d n.d. 0.27

0.032

0.001

0.14

1,063 12,081b 2.50b,c

1,828 10,278c 2.35b,c

541 2,754d 2.37b,c

1,212 16,159a 2.57b

3,434 15,023a 2.15c

886 2,852d 2.93a

259 625 0.121

< 0.001 < 0.001 0.004

< 0.001 < 0.001 0.25

18 138 52

0.46 < 0.001 0.007

1 Data are given as means and pooled SEM (untransformed data), n = 8 pens per treatment with 15 birds per pen. BD− = Basal diet without monocalcium phosphate; BD+ = Basal diet containing monocalcium phosphate; LOQ = Limit of quantification (the InsP isomer was not quantifiable in the majority of samples); n.d. = Not detectable (the InsP isomer was not detectable in the majority of samples). 2 At least one out of the following InsP3 isomers: Ins(1,2,6)P3 , Ins(1,4,5)P3 , Ins(2,4,5)P3 . 3 Means were significantly different between 500 and 12,500 FTU/kg phytase (calculated across basal diets due to missing interactions), P ≤ 0.05. 4 Means were significantly different between 0 and 500 FTU/kg phytase (calculated across basal diets due to missing interactions), P ≤ 0.05. 5 Means were significantly different between the 3 phytase levels (calculated across basal diets due to missing interactions), P ≤ 0.05. 6 Means were significantly different between 0 and 12,500 FTU/kg phytase and between 500 and 12,500 FTU/kg phytase (calculated across basal diets due to missing interactions), P ≤ 0.05. a–d Means in a row not sharing a common superscript differ significantly (multiple t-tests in case of interaction), P ≤ 0.05.

used: yij = μ + τ i + eij , where yij = jth measurement in the ith treatment, μ = overall mean, τ i = effect of the ith treatment (fixed), and eij = error term. Statistical significance was evaluated by one-way ANOVA. If the Wald-type F-test was significant (P ≤ 0.05), then differences between treatments were tested using Tukey’s honestly significant differences test at a level of significance of P ≤ 0.05.

RESULTS Supplementation and Performance Criteria During the 9-d assay period (Part 1), no significant interaction was found between MCP and phytase dosage in terms of average BW gain, FC, or feed-to-gain ratio (Table 3). Inclusion of MCP significantly increased the average BW gain (P = 0.028) and decreased the feed-to-gain ratio (P = 0.026), while phytase supplementation significantly improved all three performance criteria (P ≤ 0.001). BW gain was significantly higher and feed-to-gain ratio was significantly lower after supplementation with 12,500 FTU/kg phytase than with 500 FTU/kg (P ≤ 0.05). The highest BW gain and lowest feed-to-gain ratio followed the combination of supplemented MCP and 12,500 FTU/kg phytase. Mortality was 1.1%.

InsP6 Hydrolysis and P Net Absorption In the duodenum/jejunum (P ≤ 0.001) and lower ileum (P = 0.004), InsP6 hydrolysis was affected by a significant interaction between the MCP and phytase supplements (Table 4). In both segments, InsP6 hydrolysis was significantly lower in broilers fed BD+ without phytase compared to BD− without phytase (P ≤ 0.05). Supplementation with 500 FTU/kg phytase resulted in a significant increase in InsP6 hydrolysis in both segments in broilers fed BD− (P ≤ 0.05), but not BD+; 12,500 FTU/kg phytase caused significant further increases in InsP6 hydrolysis up to 91% in the duodenum/jejunum and 92% in the lower ileum (P ≤ 0.05). Inclusion of MCP did not alter InsP6 hydrolysis in birds fed diets with 12,500 FTU/kg phytase. A significant interaction between MCP and phytase supplements on P net absorption was detected in the duodenum/jejunum (P = 0.023), but not in the lower ileum (Table 4). This trend was the inverse of that observed for the amount of net absorbed P. In the duodenum/jejunum and in the absence of phytase supplementation, P net absorption was significantly higher for animals fed BD+ than for those fed BD− (P ≤ 0.05). Supplementation with 500 FTU/kg phytase significantly increased P net absorption irrespective of whether MCP was supplemented or not (P ≤ 0.05).

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InsP3 -a2 Ins(1,5,6)P3 Proportion of ΣInsP3 in ΣInsP3 to 5 Ins(1,2,3,4)P4 Ins(1,2,5,6)P4 3 Proportion of ΣInsP4 in ΣInsP3 to 5 Ins(1,2,3,4,6)P5 4 Ins(1,2,3,4,5)P5 5 Ins(1,2,4,5,6)P5 Ins(1,3,4,5,6)P5 Proportion of ΣInsP5 in ΣInsP3 to 5 6 ΣInsP3 to 5 5 InsP6 Pi (g/kg of DM)

BD+

0

7

PHYTATE DEGRADATION WITH PHOSPHATE AND PHYTASE Table 7. Concentrations of different InsPs and Pi in the digesta of the lower ileum.1 BD− Phytase

Pooled SEM

P-value Phy

MCP

Phy × MCP

205 25 0.034

0.90 . 0.002

0.62 . 0.67

. . 0.23

n.d. 1,030 0.39

171 355 0.043

0.48 < 0.001 0.009

0.06 0.07 0.67

0.60 0.98 0.17

177b 2,808a 1,127a 59 0.62

n.d. 191c 225c,d n.d. 0.27

67 282 108

0.002 < 0.001 < 0.001

0.37 0.026 0.001

0.023 0.016 0.023

0.059

0.013

0.17

0.40

7,183 19,159 1.72

2,166 3,623 2.05

848 1,912 0.155

< 0.001 < 0.001 0.50

0.013 0.007 0.007

0.30 0.08 0.46

500

12,500

0

500

12,500

nmol/g DM 592 n.d. 0.19

n.d. 121 0.05

n.d. < LOQ

719 < LOQ 0.16

552 < LOQ 0.07

555 166 0.34

569 n.d. 0.18

207 564 0.27

n.d. 718 0.45

766 195 0.22

496 1,965 0.31

277b 585b,c 265c,d n.d. 0.63

223b 1,098b 416c n.d. 0.68

n.d. 341c 239d n.d. 0.55

468a 996b 707b n.d. 0.62

2,289 12,888 1.33

2,629 10,672 1.30

1,299 3,749 1.30

3,851 23,404 1.83

1 Data are given as means and pooled SEM (untransformed data), n = 8 pens per treatment with 15 birds per pen. BD− = basal diet without monocalcium phosphate; BD+ = basal diet containing monocalcium phosphate; LOQ = limit of quantification (the InsP isomer was not quantifiable in the majority of samples); n.d. = not detectable (the InsP isomer was not detectable in the majority of samples). 2 At least one out of the following InsP3 isomers: Ins(1,2,6)P3 , Ins(1,4,5)P3 , Ins(2,4,5)P3 . 3 Means were significantly different between 0 and 500 FTU/kg phytase (calculated across basal diets due to missing interactions), P ≤ 0.05. 4 Means were significantly different between 0 and 500 FTU/kg phytase and between 0 and 12,500 FTU/kg phytase (calculated across basal diets due to missing interactions), P ≤ 0.05. 5 Means were significantly different between 0 and 12,500 FTU/kg phytase and between 500 and 12,500 FTU/kg phytase (calculated across basal diets due to missing interactions), P ≤ 0.05. 6 Means were significantly different between the 3 phytase levels (calculated across basal diets due to missing interactions), P ≤ 0.05. a–d Means in a row not sharing a common superscript differ significantly (multiple t-tests in case of interaction), P ≤ 0.05.

Supplementation with 12,500 FTU/kg phytase further and significantly increased P net absorption in the duodenum/jejunum (P ≤ 0.05). Inclusion of MCP did not affect P net absorption in phytase-supplemented diets. In the lower ileum, P net absorption increased after supplementation with 500 FTU/kg phytase (P ≤ 0.05), an effect that was further increased by 12,500 FTU/kg phytase (up to 71%) (P ≤ 0.05) and significantly reduced by MCP supplementation (P = 0.008). The amount of net absorbed P was increased by MCP (P ≤ 0.001) and by increasing levels of phytase in the duodenum/jejunum (P ≤ 0.05). In the lower ileum, the interaction between MCP and phytase supplements for net absorbed P (P = 0.007) was due to a significant effect of 500 FTU/kg in BD− but not BD+, and MCP having a significant effect only with 12,500 FTU/kg of phytase.

Tibia Mineralization In Part 2 of this study, values for all bone criteria were significantly higher in birds fed with 500 FTU/kg phytase in comparison birds fed without phytase (P ≤ 0.05) (Table 5). BW gain, tibia dry weight, tibia ash weight, bone breaking strength, and cortical content were further increased by supplementation with 12,500 FTU/kg phytase (P ≤ 0.05).

Appearance of InsPs and Concentrations of Pi Ins(1,2,3,4,5)P5 and Ins(1,2,4,5,6)P5 were the main InsP5 isomers detected in the small intestine. In the digesta of the duodenum/jejunum (P = 0.034) (Table 6) and lower ileum (P = 0.023) (Table 7), a significant interaction between MCP and phytase supplements was found for Ins(1,2,4,5,6)P5 concentration. Supplementation with 500 FTU/kg phytase increased the concentrations of Ins(1,2,4,5,6)P5 in animals fed BD+ (P ≤ 0.05), but not BD−. After treatment with 12,500 FTU/kg phytase, concentrations of Ins(1,2,4,5,6)P5 were low (105 to 239 nmol/g DM) and significantly lower than those measured after treatment with 500 FTU/kg phytase (P ≤ 0.05). Inclusion of MCP increased the percentage of Ins(1,2,4,5,6)P5 in ΣInsP5 (Figure 1; duodenum/jejunum: P = 0.013; in the ileum only in a trend, P = 0.07). The concentrations of Ins(1,2,4,5,6)P5 were increased by MCP in the 0 and 500 FTU/kg phytase groups (P ≤ 0.05), but not in the 12,500 FTU/kg group (Table 6 and 7). The percentage of Ins(1,2,3,4,5)P5 in ΣInsP5 significantly increased after supplementation with phytase (duodenum/jejunum: P ≤ 0.001; ileum: P = 0.001) (Figure 1). The concentrations of Ins(1,2,3,4,5)P5 were higher in the 500 FTU/kg phytase and lower in the 12,500 FTU/kg phytase groups than in the group without phytase in both

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InsP3 -a2 Ins(1,5,6)P3 Proportion of ΣInsP3 in ΣInsP3 to 5 3 Ins(1,2,3,4)P4 Ins(1,2,5,6)P4 Proportion of ΣInsP4 in ΣInsP3 to 5 4 Ins(1,2,3,4,6)P5 Ins(1,2,3,4,5)P5 Ins(1,2,4,5,6)P5 Ins(1,3,4,5,6)P5 Proportion of ΣInsP5 in ΣInsP3 to 5 5 ΣInsP3 to 5 6 InsP6 5 Pi (g/kg of DM)

BD+

0

8

ZELLER ET AL.

segments (P ≤ 0.05) (Tables 6 and 7; in the ileum in the BD− group, this trend was not significant). However, in the lower ileum, a significant interaction between MCP and phytase supplements affected the concentrations of Ins(1,2,3,4,5)P5 (P = 0.016); this interaction was due to significantly higher concentrations of Ins(1,2,3,4,5)P5 in the BD+, 500 FTU/kg phytase group than in the BD−, 500 FTU/kg phytase group (P ≤ 0.05). The concentrations of Ins(1,2,3,4,6)P5 were increased after inclusion of MCP (P ≤ 0.025) and reduced by supplementation with phytase in the duodenum/jejunum (P = 0.001) (Table 6). The percentage of Ins(1,2,3,4,6)P5 in ΣInsP5 declined with increasing phytase doses in both segments

(P ≤ 0.001) (Figure 1). Ins(1,3,4,5,6)P5 was nearly undetectable in the small intestine, with the exception of a low concentration in the ileum of birds fed BD+ with 500 FTU/kg phytase. Ins(1,2,5,6)P4 was detected after treatment with phytase in the duodenum/jejunum; levels were significantly higher after treatment with 500 FTU/kg phytase compared to 12,500 FTU/kg phytase (P = 0.042) (Table 6). In the lower ileum, the concentrations of Ins(1,2,5,6)P4 were significantly increased by supplementation of phytase (P ≤ 0.001) (Table 7). Inclusion of MCP significantly increased the concentrations of Ins(1,2,5,6)P4 in the duodenum/jejunum (P = 0.006) and tended to

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Figure 1. InsP5 isomers in the digesta of the duodenum/jejunum (A) and ileum (B), expressed as percentage of ΣInsP5. Values are mean ± SE (untransformed data; n = 8 pens/treatment, 15 birds each). BD−, basal diet without monocalcium phosphate; BD+, basal diet containing monocalcium phosphate.

PHYTATE DEGRADATION WITH PHOSPHATE AND PHYTASE

DISCUSSION InsP6 Hydrolysis and P Net Absorption High InsP6 hydrolysis (67%) and P net absorption (52%) were measured in the lower ileum for animals fed the BD− diet, which contained no detectable phytase activity or mineral P. These data agree with previous results and illustrate the strong potential of broilers and their gut microbiota to hydrolyze InsP6 in low-P and low-Ca diets (Tamim and Angel, 2003; Leytem et al., 2008; Shastak et al., 2014; Zeller et al., 2015). Despite this high basal level of hydrolysis, supplementation with 500 FTU/kg phytase increased InsP6 hydrolysis in animals fed BD−, and the 12,500 FTU/kg of phytase led to InsP6 hydrolysis and P net absorption of 92 and 71%, respectively. Our observation of 91% InsP6 hydrolysis in the duodenum/jejunum with the 12,500 FTU/kg phytase dosage suggests rapid InsP6 hydrolysis in the upper digestive tract, with better phytate solubility and pH conditions for the supplemented phytase than in the lower gut. However, even very high amounts of phytase (12,500 FTU/kg) did not cause complete InsP6 hydrolysis. Since no differences in InsP6 hydrolysis were found between intestinal segments for any treatments with 12,500 FTU/kg phytase, the remaining substrate was probably not accessible. The remaining 8% of feed InsP6 may have been nonextractable even in the acidic regions of the digestive tract. Short retention time in the acidic anterior segments followed by precipitation in the intestinal segments could also underlie the observed incomplete InsP6 hydrolysis. Previous reports documented the dependency of InsP6 hydrolysis, without or with supplemented phytase, on Ca and P levels (Manangi and Coon, 2008; Delezie et al., 2012). The present study also showed lower InsP6 hydrolysis for BD+ compared to BD−, both without phytase. The significant interaction between MCP and phytase that affected InsP6 hydrolysis demonstrates that MCP is critical to the effect of added

phytase. Supplementation with 500 FTU/kg phytase did not significantly increase InsP6 hydrolysis in animals fed BD+, but did in those fed BD−. This MCP effect could be related to a reduction in the activity of phytases from the microbiota or mucosa due to inhibition by the end product Pi . Decreased endogenous mucosal or microbiota-associated phytase may also result from the down-regulation of phytase expression or from a shift in the microbial population to fewer phytaseproducing organisms. However, inclusion of MCP had no significant effect on activity of mucosal phytase measured in isolated jejunal brush border membrane preparations in broilers from this study fed diets without or with 500 FTU/kg of phytase (Huber et al., 2015). Alternatively, the higher Ca:InsP6 ratio in BD+ groups compared to BD− groups may lead to increased generation of insoluble, undegradable Ca-InsP6 precipitates at the pH of the small intestine (Applegate et al., 2003). In groups fed the diets supplemented with 500 FTU/kg, it is not clear whether the observed MCP-mediated reduction in InsP6 hydrolysis was related to the aforementioned effects or to MCP’s inhibition of the activity of the added phytase. However, MCP did not affect InsP6 hydrolysis at the high phytase level, which confirms one hypothesis of this study. This study provides the first confirmation of the interaction between enzyme and Pi supplementation for phytase supplementation in broilers. The concentrations of liberated InsP-Pi and Pi from MCP were likely too low to trigger relevant product inhibition at the high concentration of phytase. As a consequence, in the presence of 12,500 FTU/kg phytase, more P was net absorbed by animals fed BD+ than by animals fed BD−. Hence, in contrast to standard phytase levels, high doses of phytase and mineral P exerted additive effects on the amount of net absorbed P in the intestine. This additive effect also manifested in larger BW gain and a lower feed-to-gain ratio for animals fed BD+ compared to those fed BD− at 12,500 FTU/kg phytase. Further, the higher growth and tibia mineralization following treatment with 12,500 FTU/kg phytase (Tables 3 and 5) confirmed that additional P was released, absorbed, and deposited by the broilers. Whether higher levels of mineral P show such additive effects with high doses of phytase still needs to be clarified.

Appearance of InsPs as affected by Supplemented Phytase One objective of this study was to investigate the effect of various phytase dosages on the concentrations of InsPs in the small intestine. Most likely, the appearance of InsPs results from the combined effects of supplemented phytase in addition to phytases and other phosphatases of microbial and mucosal origin. In a previous study, Ins(1,2,3,4,5)P5 and Ins(1,2,5,6)P4 (perhaps co-eluted with Ins(2,3,4,5)P4

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increase the concentrations of Ins(1,2,5,6)P4 (P = 0.07) and Ins(1,2,3,4)P4 (P = 0.06) in the lower ileum (Tables 6 and 7). In the presence of phytase, InsP3 -a was only detected in both segments in the animals who received MCP supplementation. A significant interaction between MCP and phytase supplements was found for concentration of Pi in the duodenum/jejunum (P = 0.007), but not in the lower ileum (Table 6 and 7). When MCP was supplemented concentrations of Pi were significantly lower in the 500 FTU/kg and significantly higher in the 12,500 FTU/kg phytase groups than in the other 2 phytase groups in the duodenum/jejunum (P ≤ 0.05) (Table 6). In the lower ileum, the concentrations of Pi were increased after inclusion of MCP (P = 0.007) (Table 7). Pearson correlation coefficients showed no significant values between concentrations of Pi and the amount of net absorbed P.

9

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Effect of Supplemented MCP on the Appearance of InsPs without or with Supplemented Phytase The InsP pattern observed in the intestine is indicative of the types of phosphatases involved in InsP6 breakdown and the effects of MCP and the added phytase. Upon MCP supplementation, the concentrations of ΣInsP3 to 5 in the digesta increased (Tables 6 and 7), suggesting less degradation of lower InsPs as well as less InsP6 hydrolysis in the presence of MCP. Higher concentrations of ΣInsP3 , ΣInsP4 , and ΣInsP5 were also reported by Agbede et al. (2009) in the excreta of cecectomized hens fed a high-P diet compared to a low-P diet. In the present study, the proportions of ΣInsP3 , ΣInsP4 , and ΣInsP5 in ΣInsP3 to 5 were

not affected by MCP. Hence, although there was no inhibition of a single degradation step in the breakdown process, there was a change in the InsP pattern within isomers of the same degree of phosphorylation when MCP was provided as a supplement. In both segments of the intestine, a higher percentage of Ins(1,2,4,5,6)P5 in ΣInsP5 (ileum: P = 0.07) occurred in the presence of MCP, and the concentrations of Ins(1,2,4,5,6)P5 were higher for animals fed BD+ than for animals fed BD−, both without phytase. The concentrations of InsP3 a [Ins(1,2,6)P3 ] tended to be higher for BD+ without phytase compared to BD− without phytase, and Ins(1,2,5,6)P4 additionally appeared in the ileum of animals fed BD+ without phytase. These three isomers can be produced by 3-phytase (Greiner et al., 2009). The inclusion of MCP also reduced the percentage of Ins(1,2,3,4,5)P5 in ΣInsP5 in the duodenum/jejunum (Figure 1A), whereas in the ileum, the percentage of Ins(1,2,3,4,6)P5 in ΣInsP5 was reduced by MCP. These changes in the InsP pattern demonstrate that supplemental MCP decreased or increased the activities or expression of specific microbiota-associated or mucosal phytases and other phosphatases, producing different InsPs, or caused a shift in the microbial population. Activity of 3-phytase has been detected in several bacteria, such as Enterobacter cloacae or species of Selenomonas, Bacillus, Pantoea, and Klebsiella (Greiner et al., 1997; Greiner et al., 2002; Greiner, 2004; Herter et al., 2006; Puhl et al., 2007). When MCP was provided as a supplement in the present study, the levels of the main degradation product of 6-phytase were reduced in the duodenum/jejunum, whereas the levels of the main degradation product of 5-phytase were reduced in the ileum. Abudabos (2012) demonstrated a decrease in mucosal phytase activity in chickens with higher dietary tP. Huber et al. (2015) found reduced mucosal phytase activity close to the level of significance when MCP was supplemented in combination with 12,500 FTU/kg phytase, indicating reduced mucosal phytase expression due to high amounts of luminal Pi . In vitro, phytate-degrading enzyme activity or synthesis was depressed by available Pi in several microorganisms (Greiner et al., 1997; Shieh et al., 1969). Metzler-Zebeli et al. (2010) observed a change in the ileal bacterial population following the administration of supplemental MCP in pigs. In general, the composition of the microbial community can be affected by MCP, since bacteria are thought to differ in their P requirements (Komisarczuk et al., 1987) and in their ability to produce phytase. Ca also was reported to affect ileal bacterial populations in pigs, the phytase activity of specific bacteria in vitro, and the mucosal phytase of chickens (Abudabos, 2012; MetzlerZebeli et al., 2010; Shimizu, 1992). However, we cannot separate the effects of P and Ca in the present study. As discussed previously for InsP6 , the precipitation of Ca–InsP complexes may also influence the degradability of the lower InsPs. If so, then a similar influence on the different positional InsP isomers would

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because enantiomers were not separated) were specified as the main InsP4 to 5 isomers formed in broilers following supplementation with the same phytase product used in the present investigation (Zeller et al., 2015). This observation was confirmed by the present data, as the percentage of Ins(1,2,3,4,5)P5 in ΣInsP5 and concentrations of this InsP5 isomer were higher after exposure to 500 FTU/kg phytase than without phytase, and concentrations of Ins(1,2,5,6)P4 were significantly increased by supplementation of phytase. To the best of our knowledge, only one published study in pigs and one in broilers reported the dependency of InsP degradation on the level of supplemented phytase (Kemme et al., 2006; Walk et al., 2014). In the pig study, InsP6 hydrolysis was higher after supplementation with 900 FTU/kg than with 150 FTU/kg, and InsP6 was rapidly degraded to InsP3 and InsP2 with 900 FTU/kg (Kemme et al., 2006). In contrast, with 150 FTU/kg the small intestine hosted a broader spectrum of intermediate InsPs, such as InsP4 (Kemme et al., 2006). In the recently published broiler study (Walk et al., 2014), differences in the levels of InsP3 and inositol were found in the gizzard with increasing dosages (200, 1,000, and 1,500 FTU/kg) of the same phytase used in the present study. In the present study, low concentrations of Ins(1,2,3,4,5)P5 and Ins(1,2,4,5,6)P5 followed supplementation with 12,500 FTU/kg phytase, suggesting more complete degradation of InsP5 at the higher phytase dosage; at the lower dosage, InsP5 was the predominant InsP. For treatments with 12,500 FTU/kg phytase, Ins(1,2,5,6)P4 was the predominant InsP, and it was degraded more completely than after 500 FTU/kg phytase supplementation in the duodenum/jejunum but not in the lower ileum. However, stronger degradation of InsP5 to 6 is relevant to the solubility and degradability of InsPs in total, since InsP3 and InsP4 have lower, though still relevant, mineralbinding strength (Xu et al., 1992; Luttrell, 1993; Persson et al., 1998). This strong InsP5 to 6 degradation resulted in high P net absorption following treatment with 12,500 FTU/kg phytase.

PHYTATE DEGRADATION WITH PHOSPHATE AND PHYTASE

without or with standard levels of phytase. This observation indicates a lower susceptibility of high-dose phytase in combination with MCP. Further studies should identify the phytase and mineral P levels that maximize InsP degradation and increase the amount of absorbed and retained P.

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be possible. However, the aggregation potential of Ca may differ for different positional InsPs, as reported for protein and iron (Hawkins et al., 1993; Yu et al., 2012), which may explain the effect of MCP on specific InsPs. Further studies are needed to separate the effects of Ca and P and to investigate the influence of these elements on microbial populations and mucosal phytases. The interactions between MCP and supplemental phytase on specific lower InsPs imply that the effect of MCP is dependent on the activity of supplemental phytase, as shown for InsP6 hydrolysis. Ins(1,2,3,4,5)P5 and Ins(1,2,5,6)P4 occurred in significantly or borderlinesignificantly higher concentrations in animals fed BD+ than in animals fed BD−, both with 500 FTU/kg phytase. Thus, degradation of the main hydrolysis products of phytase continued to a lesser extent in the presence of MCP at the standard phytase dosage. As discussed previously regarding InsP6 hydrolysis, the present study cannot distinguish whether this effect was related to a change in the activity and composition of phosphatases present in the intestine, to changes in supplemented phytase activity, or to both, as a consequence of MCP supplementation. As hypothesized here, MCP combined with 12,500 FTU/kg phytase had no significant effect on the concentrations of the lower InsPs, as shown for InsP6 hydrolysis. This observation affirms that higher doses of phytase are less affected than lower doses at the MCP level used here. However, although the concentrations of InsP5 isomers were not influenced by the inclusion of MCP at 12,500 FTU/kg phytase, the concentrations of Ins(1,2,5,6)P4 and InsP3 a [Ins(2,4,5)P3 ], also described as a degradation product of an E. coli phytase (Greiner et al., 2000), tended to be higher in both intestinal segments of animals fed BD+ than in animals fed BD−, both with 12,500 FTU/kg phytase. This observation suggests a slight influence of MCP (at the level used) on the degradation of lower InsPs when birds were fed diets with such high phytase dosage. We conclude that the initial step in InsP6 hydrolysis is not the only step in the breakdown process that is influenced by supplementation with MCP or phytase. Both factors exerted a selective influence on specific InsPs, perhaps due to variations in this influence on the activities of specific phosphatases in the intestine. Further studies are needed to investigate the effects of supplementation on the microbial population and on the mucosal phosphatases. Interactions between MCP and phytase affected InsP6 hydrolysis and specific InsPs, demonstrating that MCP is critical to the efficacy of supplemented phytase. Applying phytase doses that are several-fold higher than the industry standard caused >90% hydrolysis of feed InsP6 and stronger degradation of InsP5 , resulting in higher P net absorption than at the standard phytase dose. Supplements of MCP used at 12,500 FTU/kg phytase did not affect InsP6 hydrolysis or InsP5 degradation, but lowered InsP6 hydrolysis and weakened the degradation of lower InsPs in diets

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