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A W Jongbloed, Z Mroz and P A Kemmeacid in different sections of the alimentary tract.

concentration and apparent digestibility of dry matter, total phosphorus, and phyticThe effect of supplementary Aspergillus niger phytase in diets for pigs on

1992, 70:1159-1168.J ANIM SCI

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The Effect of Supplementary Aspergillus nigerPhytase in Diets for Pigs on Concentrationand Apparent Digestibility of Dry Matter,Total Phosphorus, and Phytic Acidin Different Sections of the Alimentary Tract'A. W. Jongbloed, Z. Mroz, and P. A. Kemme

Research Institute for Livestock Feeding and Nutrition,Lelystad, The Netherlands

ABSTRACT Six barrows of approximately 37 kgBW, fitted with two simple T-cannulas in theduodenum (25 cm posterior to the pylorus) andterminal ileum (12 to 15 cm anterior to theileocecal junction), were fed two diets containing2.1 g of P/kg in the form of phytic acid an d a lowintrinsic phytase activity (comsoybean mealbased diet [Diet AI or a typical Dutch diet [Diet BIwithout or with supplementary microbial phytasefrom Aspergillus niger (var. ficuuml equal to 1,500phytase units per kilogram of diet, in a crossoverdesign. The apparent duodenal, ileal, and totaltract (overall) digestibilities of DM, total P, andphytate P (phytic acid x .282)were calculated usingboth Cr-NDR (neutral detergent residue mor-danted with Cr) and Co-EDTA as dual-phase

markers. Concentration of tota l P in the ilealdigesta (P e .01) and feces P c .0011 of pigs fedmicrobial phytase was lower than without thisenzyme, irrespective of the diet. Ileal digestibilityof total P was 18.5 and 29.8 percentage unitshigher (which was a 1.7-o 2.9-fold increase) due toadded Aspergillus niger phytase P e .05). Also,total t ract (overall) digestibility increased by 27.0to 29.7 percentage units (P e .011. Phytic acidconcentration in the duodenal and ileal digesta ofpigs receiving microbial phytase was lower P e.01or .0011, resulting in its higher ileal digestibility(dephosphorylation rate) by 50.1 percentage unitsfor Diet A and by 75.4 percentage units for Diet B.Irrespective of the treatment, no phytase activitycould be detected in the ileal digesta of pigs.

Key Words: Pigs, Cannulation, Phytase, Phosphorus

IntroductionSupplementary microbial phytase from Asper-gilli in diets for pigs is used to enhance digestibil-ity of myo-inositol phosphates, insoluble com-plexes of phytic acid, and minerals from feedstuffsof plant origin (Han, 1989; Zyla et al., 1989; Simonset al. 1990). A degree of phytate degradation(dephosphorylation, hydrolysis) may also berelated to the presence of intrinsic plant phytases,

'The authors wish to thank the Gist-brocades The Nether-lands) for financial support, all the coworkers at the IVVOCLelystad), CIVO Zeist), Gist-brocades Delftl, and he Phospho-rus Working Group from the Commodity Board for Feedstuffs(The Netherlands) for their excellent advice.Received January 25, 1991.Accepted October 31, 1991.

J. Anim. Sci. 1992. 703159-1168

phytases from the bacterial flora in the gut, andintestinal mucosal phytases Williams and Taylor,19851, although Pointillart (1988) concluded thatintestinal phytase activity in pigs is negligible.According to Cosgrove (19801, Aspergilli produce3-phytase (EC 3.1.3.81, a nonspecific phos-phomonoesterase, catalyzing the following reac-tion: myo-inositol hexakisphosphate H20 + D-myo-inositol - 1,2,4,5,6-pentakisphosphate or-thophosphate. This reaction proceeds in a step-wise manner, producing five classes of intemedi-ate products (myo-inositol pentakis-, tetrakis-, tris-,bis-, and monophosphates) of variablestereochemistry (Maga, 1982; Frnrlich et al. 1986).However, the effect of this enzyme on phytatedegradation in the gastrointestinal tract of pigs isstill not well elucidated and in vitro studies of Hanand Wilfred (19881, Han (19891, and Bos (19881 do

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1160 JONGBLOED ET AL.not necessarily relate quantitatively to the in vivoconditions, in which factors such as gastric empty-ing, retention time, variation of pH and digestacomposition, and interactions between mineralsand other nutrients (dietary and endogenous) mayaffect the dephosphorylation reactions. Besides,myo-inositol phosphate is capable of forminginsoluble complexes containing Ca and othercations, and the extent to which such complexesare formed in vivo in animals fed practical diets isdependent on the concomitant presence of highconcentrations of each constituent in the intestinalchyme (Oberleas and Moody, 1982; Suttle, 1983).Therefore, the present experiment was conductedto study the effect of Aspergillus niger bar. icuumlphytase in diets for pigs on the concentration andapparent digestibility of DM, total P, and phyticacid in different sections of the alimentary trac t.

Materials and MethodsAnimaZs. Six barrows from a three-breed rota-tional cross (Yorkshire, Finnish Landrace, andDutch Landrace) of 37-kg average in itial BW, fittedwith two simple T-cannulas under inhalationanaesthesia (one located in the duodenum approx-imately 25 cm posterior to the pylorus and anotherin the terminal ileum, approximately 12 to 15 cmanterior to the ileocecal junction), were used inthis experiment. After a 14-d recovery period, thepigs were housed individually in pens of 2.00 m x

1.45 m, at ambient temperature (18OC1, and assigned to four experimental treatments (providingsix replications) according to a crossover design asfollows: 1) corn-soybean meal diet (Diet A withoutmicrobial phytase, 21 corn-soybean meal diet (DietA) with microbial phytase, 3) typical Dutch diet(Diet B)without microbial phytase, a nd 41 typicalDutch diet (Diet B) with microbial phytase. FinalBW of the animals was approximately 75 to 80 kg.Diets. The major components of Diet A wereground corn a nd extracted soybean meal, whereasDiet B contained tapioca, extracted soybean meal,hominy feed, extracted sunflower meal, and soy-bean oil (Table 1 . These components are known tohave a low intrinsic phytase activity ( e 5 phytaseunits per kilogram of feed). Both diets werecalculated to have similar protein (approximately11.5%digestible protein) and energy levels (approx-imately 12.5 MJ of ME/kg or 3,000 kcal of ME/kg).Treatments 2 and 4 were supplemented with 4 g /kg of crude microbial phytase preparation fromAspergillus niger (var. f icuum) (equal to approxi-mately 1,500 phytase units/kg of diet). This en-zyme was obtained from A. niger (var. ficuurn)strain NRRL 3135 according to the proceduredescribed by Simons et al. (1990). The activity ofthe crude microbial phytase showed pH optima at

pH 5.5 and 2.5. The enzyme was able to hydrolyzephytate complexes in vitro in soybean meal, corn,and liquid compound feed for pigs. The thermalstability of the microbial phytase was good &e.,95% of phytase remained active when a meal wassteam-heated at 50C, as part of the pelletingprocess). Pigs were fed twice daily (0700 and 1500)in a wet, mash form at a feeding level of 2.3 timesmaintenance requirement (maintenance require-ment = 418 kJ of ME/BW.75). Daily rations weresupplemented with 4 g of neutral detergent resi-due mordanted with Cr per kilogram of diet as anindigestible marker for the solid phase and Co-EDTA (5 g/kg of diet) for the liquid phase andmixed with water at a ratio of 2.5:l (vol/wt) justbefore feeding. The pigs had no access to waterbetween feedings.Collection Procedures. Each treatment was testedfor 14 d. After the first 3 d, samples of duodenaldigesta were collected five times at 1- or1.5-h intervals after the morning meal on the 4thand 6th d of the test period. Feces were collectedat random on the 9th and 10th d, whereas samplesof ileal digesta were collected quantitatively onthe loth, Wth, and 14th d (seven times in1- to 2-h intervals, beginning at 0700).The samplesof digesta were collected into sterilized polyethyl-ene bags attached to the cannula barrel and werefrozen immediately after the pH was measured.

Table 1. Ingredients and nutritive valueof experimental dietsDiet A Diet B(corn-soybean (TypicalItem meal) Dutchl

Ingredient g/kgCorn 859.50 -Soybean meal 124.55 124.55Tapioca - 421.00Hominy feed - 336.00Sunflower meal - 80.00Soybean oil - 26.00Limestone 11.80 8.30Salt 2.50 2.50Trace mineral-vitamin premix* 1.40 1.40Choline chloride .25 .25GE,M J k g 15.9 16.2ME,MJ/kg 12.7 12.4

Calculated nutritive value

Digestible protein, g/kg 114 119Digestible P, g k g .9 1.2Digestible lysine, g/kg 5.0 5.0

%ace mineral-vitamin premix contained the following inml l igr m per kilogram of diet: 2.4 (8,000 IU) itamin A .04(1,600U) vitamin D3, .0 (8.0 IU) itamin E, 4.0 riboflavin, 20.0nicotinic acid, 8.0 panthotenic acid, .02 vitamin BIZ,125.0 antiox-idant (4 to 5% BHA, 4 to 5% ethoxyquin, 4 to 5% citric acid, 2 to3% o-phosphoric acid, 2 to 3% E 471 fatty acid, and Si02 ascarrier), 50 MnO, 155 %SO4, 4 0 CuS04 2 KI, 430 FeS04, .3 Se,and 555.5 carrier.

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ASPERGILLUS NIGER PHYTASE IN DIETS FOR PIGS 1161Thereafter, they were freeze-dried and ground topass a 1-mm sieve before analysis.Analytical Procedures. Dry matter was deter-mined after freeze drying and nitrogen contentwaa determined by the Kjeldahl method [AOAC,1080). Crude protein was calculated as Kjeldahl Nx 6.25. Content of Ca, Mg, Na, K, Cr, and Co wasassayed by atomic absorption spectrophotometryafter ashing the samples a t 46OOC and preparingmineral solutions in .48 M HC1. Total P was deter-mined colorimetrically by the vanadomolybdateprocedure (AOAC, 10801. Phytic acid was extractedand quantitatively assayed by the HPLC methodof Simons et al. (10001, whereas separation of myo-inositol tetrakis- and pentakisphosphates fromdegraded phytates was carried out using an anion-exchange column (Dionex, AS 3, Dionex, Sun-nyvale, CAI with OO M H N 0 3 at a flow rate of 1.0ml/min IBos, 1088). Measured concentrations ofparticular nutrients in the samples of digesta fromthe duodenum an d the ileum of the experimentalpigs were related to the tota l volume of digestapassing these sections within the time of oursampling by an extrapolation from the pattern offlow of digesta described by Braude et al. (10761.Phytase activity in the diets was assayed bymeasuring the amount of o-phosphates releasedfrom phytic acid within a period of linear increasewith time (Simons et al. 1990). One unit of phytase

activity is equal to 1 p o l of o-phosphate liberatedfrom 1 m o l of phytic acid within 1 min at 40Cand pH 5.5.Statistical Analysis. Each pig was the experi-mental unit. All data were statistically analyzedusing the analysis of variance according to thewhere N = overall mean, A = effect of animal, P= effect of period, T = effect of treatment, and e= error contribution with average 0 and variance0 2 , a n d i = l . . . a , j = l . . . b , k = l .. . n.T he re wa sno evidence of a P x T interaction. Treatmentmeans were compared by the Student t-test at P