Biodisponibilidad Del Zn Org e Inorg

7/27/2019 Biodisponibilidad Del Zn Org e Inorg

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K J Wedekind, A J Lewis, M A Giesemann and P S Millermeal diets.

Bioavailability of zinc from inorganic and organic sources for pigs fed corn-soybean

1994, 72:2681-2689.J ANIM SCI

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Bioavailability of Zinc from Inorganic and Organic Sources for PigsFed Corn-Soybean Meal Diets1f2f3

K. J. Wedekind4, A. J. Lewis5, M. k Giesemann, and P. S. MillerDepartment of Animal Science, University of Nebraska, Lincoln 68583-0908

ABSTRACT: Two experiments were conductedwith pigs 1) t o determine the effect of supplementalZn on growth performance, bone Zn, and plasma Zn inpigs fed Zn-unsupplemented, corn-soybean meal dietsand 2) to assess bioavailability of Zn from inorganicand organic Zn sources. In both experiments , weanlingpigs were fed a diet with no supplemental Zn fo r 5 wkt o deplete their Zn stores. In Exp. 1, 192 pigs were feda corn-soybean meal diet (growing diet, 32 mgk g ofZn; finishing diet, 27 mg kg of Zn) supplemented withfeed-grade ZnS04.H20 t o provide 0, 5 , 10, 20, 40, and80 mgkg of supplemental Zn. Supplemental Zn didnot affect weight gain, feed intake , o r gaidfeed duringeither the growing or the finishing period ( P > .05).However, bone and plasma Zn concentrations in-creased linearly (P < O 1 in response to supplementalZn at dietary Zn levels between 27 mgkg (b as al ) and

47 mgkg (breakpoint). In Exp. 2, three levels ofsupplemental Zn from ZnS04.H20 (0 , 7 .5 , and 15 mg/kg of supplemental Zn) were used t o construct astandard curve (metacarpal, coccygeal vertebrae, andplasma Zn concentrations regressed on supplementalZn intake; R2 = .93, .89, and .82, respectively). Fromthe standard curve, the bone and plasma Zn concen-trations obtained from pigs fed 15 mgkg of sup-plemental Zn from ZnO and 7.5 mgkg of supplementalZn from Zn-methionine (ZnMET) and Zn-lysine(ZnLYS) were used t o calculate bioavailable Zn viamultiple linear regression, slope-ratio analysis. Theestimates of Zn bioavailability differed depending onwhich variable was used. Overall trends indicated thefollowing rankings: ZnS04.H20 > ZnMet > ZnO >ZnLys.

Key Words: Pigs, Zinc, Bioavailability, Zinc Methionine, Zinc Sulfate, Zinc Oxide

IntroductionZinc bioavailability studies have traditionally beencarried out with semipurified diets containing egg

white, casein, o r soy isolate as the protein source. Useof semipurified diets with pigs is very expensive.Furthermore, recent experiments with chicks(Wedekind et al., 1992) have demonstrated that therelative bioavailability estimates determined amongZn sources differed substantially depending onwhether the basal diet used was a purified (crystal-line amino acid), soy isolate, or a corn-soybean mealdiet.

lJournal series no. 10555, Agric. Res. Div., Univ. of Nebraska.2Partial financial support by Zinpro Corporation, Chaska, MN

55318 is gratefully acknowledged.3The assistance of D. Oberleas (Texas Tech Univ .) in analyzing

phytate and the technical assistance of R. M. Diedrichsen and J. L.

J. h i m . Sci. 1994. 72:2681-2689

Previous studies with pigs (Hill et al., 1986;Swinkels et al., 1991) have failed t o show differencesin Zn bioavailability between organic (complexes o rchelates) and inorganic Zn sources. Yet, markeddifferences in bioavailability have been documented inpoultry diets (Wedekind et al., 1992). The datareported by Wedekind et al. (19 92 ) indicated that , forchicks fed corn-soybean meal diets, Zn from a Zn-methionine complex (ZnMET) was 206% bioavailablerelative to a ZnS04.H20 standard (i.e., l o o%) ,whereas ZnO provided only 61% bioavailable Zn.

The purpose of the research reported herein was 1)to determine whether a corn-soybean meal diet couldbe used t o assess Zn bioavailability in pigs and 2) t ocompare the bioavailabil ity of Zn from ZnMET, Zn-lysine (ZnLYS) , and ZnO relative t o ZnSOq.Hz0.

Materials and MethodsKovar is gratefully acknowledged.Topeka, KS 66601.

Animals , Pretest Period, and Housing. Two hundredforty weanling pigs (Yorkshire x Landrace x Hamp-shire x Duroc; weaned at 28 d ) were given ad libitumaccess to a star ting diet (20% CP ) with no supplemen-tal Zn (Table 1 ) for 3 wk. Pigs were then switched t o

lPresent address: Mark Morris Associates, P. 0. Box 1658,5T0 whom correspondence should be addressed.Received December 3, 1993.Accepted June 24, 1994.

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2682 WEDEKIND ET AL .an 18% CP starting diet (Table l ) , also with nosupplemental Zn, fo r the next 2 wk. Our objective wast o deplete Zn stores during this pretest period. Therewere six pens of barrows and six pens of gilts, eachcontaining 20 pigs per pen. The nursery pens hadpainted concrete walls and plastic-coated wire floor-ing. Feeders were constructed of stainless steel.Water, supplied from stainless steel nipple waterers,contained no detectable Zn. The 20 and 18% CPstarting diets were analyzed for Zn and found t ocontain 42 and 37 mgkg, respectively. The NRCrequirements for Zn (NRC, 1988 ) for pigs tha t weigh5 to 10 kg and 10 t o 20 kg are 100 and 80 mgkg,respectively. The protocol during the pretest periodwas identical for Exp. 1 and 2.

From these 240 pigs, 192 pigs were allotted t o sixdietary treatments. There were four replicates pertreatment with eight pigs per pen. The pigs wereblocked by sex such that there were 12 pens ofbarrows and 12 pens of gilts. Pigs were also blocked byweight such that, within sex, pigs were randomlyallotted across treatment. Within each block, eachtreatment had a similar mean initial weight andweight distribution. Pigs were housed in a modified-open-front building with pen sizes of 1.8 m x 3.6 m.Pens were constructed of 50% solid and 50% slattedfloors. Twelve of the pens contained painted wire inthe slatted area and the other 12 pens had plastic-coated wire. Pen partitions were solid concrete and the

end-gates were metal. All galvanized and non-concretesurfaces were painted. Pigs were allowed ad libitumaccess to feed and water from a four-hole woodenfeeder and a stainless steel nipple waterer. The pigsaveraged 25 kg (Exp. 1) and 20 kg (Exp. 2) at thebeginning of the growing period and were fed thegrowing diets for 6 wk. Pigs were then switched tofinishing diets (pig s averaged 55 kg in Exp. 1 and 50kg in Exp. 2) and were maintained on these diets(14% CP and the same Zn supplement as in thegrowing period) until the replicates averaged approxi-mately 100 kg. The final weight of the pigs was 104 kgin Exp. 1 and 97 kg in Exp. 2. Experiment 1 wasconducted from January through April and Exp. 2 wasconducted from July t o November. Pig weights andfeed consumption were measured every 2 wk.Diets and Experimental Treatments. A corn-soybeanmeal basal diet (Table 1 ) was formulated t o beadequate in all nutrients except Zn (NRC, 1988).Dietary additions of Zn were made at the expense ofCaC03 in the mineral mix. The mineral mix con-stituted .l% of the diet. In Exp. 1, the basal diet wassupplemented with feed-grade Z n S 0 4 - H 2 0 t o provide0, 5 , 10, 20, 40, and 80 mgkg of supplemental Zn.Total dietary Zn levels in the growing diets rangedfrom 32 to 112 mgkg. Phytate:Zn molar ratios rangedfrom 12.3 t o 2.6 and (Ca x phytate):Zn molar ratiosranged from 1.8 t o .37. The Zn levels in the finishingdiets ranged from 27 to 107 mgkg, which cor-responded to phytate:Zn molar ratios that ranged from

Table 1. Composition of diets (Exp. 1 and 2)aStarting Growing Finishing

Ingredient, lo (20% CP) (18% CP) (16% CP) (14% CP)CornSoybean meal (44% CP)Dried edible wheyOatsTallowDicalcium phosphateLimestoneSaltVitamin premixbZn-free mineral mixCChlortetracycline mixdAnalysis

CP, %Ca, %P, %Zn, mg/kgPhytate, mp/g

40.8032.3515.005.003.001.40.05.30

1.00.10

1.0020.7

.76

.683.71

42

56.3527.0010.003.001.05.20.301oo.10

1.00

-

17.9.78.61

3.3837

70.1023.25--4.001.10.45.30.70.10-

16.1.65.51

2.9032

76.0517.45--4.001.00.40.30.7 0.10-

14.4.58.48

3.6427

aAs-fed basis.bProvided the following per kilogram of premix: retinyl acetate, 440,920 IU; cholecalciferol, 55,115 IU;all-ruc-a-tocopherol acetate, 2,205 IU; menadione, 331 mg provided as menadione sodium bisulfatecomplex; niacin, 3,307 mg; riboflavin, 551 mg; cyanocobalamin, 2.2 mg; D-pantothen ic acid, 2,205 mg as D-calcium panto thenate; choline, 11.0 g as choline chloride; ethoxyquin, 100 mg; CaC03, 550 g; rice hulls,404 g.'Provided the following per kilogram of premix: Ca, 117.46 to 198.27 g as CaC03; Cu, 11.02 g asCuS0 46H2 0; I, .22 g as Ca(IO3)2.H20; e, 110.23 g as FeS04.HzO; Mn, 22.05 g as MnO; Se, .30 g asNa Se03.%Chlortetracycline mix added a t 110 mg/kg.by guest on February 25, 2013www.journalofanimalscience.orgDownloaded from


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ZINC BIOAVAILABILITY FO R SWINE 268314.7 t o 3.4 and ( C a x phytate):Zn molar ratios thatranged from 2.1 t o .47. In Exp. 2, three levels of Znfrom feed-grade ZnS04.HzO ( 0, 7.5, and 15 mg/kg ofsupplemental Zn) were used t o construct a standardcurve (bone and plasma Zn concentration regressed onsupplemental Zn intake). From the standard curve,the bone and plasma Zn concentrations obtained frompigs fed ZnO, ZnMET, and ZnLYS were used tocalculate the bioavailability of Zn in these diets bymultiple regression, slope-ratio analysis. The ZnO wasadded to provide 15 mgkg of supplemental Zn, andZnMET and ZnLYS were added to provide 7.5 mgkgof supplemental Zn. The Zn sources that wereperceived to be more available than the standard weretested at an intermediate level of Zn t o ensure thatbone and plasma Zn levels fell in the linear responserange. Previous studies with chicks (Wedekind e t al.,199 2) had indicated tha t ZnO was less available,whereas ZnMET was more available, thanZnS04.H20. The feed-grade sources of ZnS04.HaO andZnO were provided by Southeastern Minerals Corp.(Bainbridge, GA); ZnMET and ZnLYS were providedby Zinpro Corp. (Chas ka, MN).The diets were analyzed fo r CP, Ca, P, Zn, andphytate. Crude protein ( N x 6.25) was determinedusing Kjeldahl procedures described by AOAC (1984).Diets were analyzed for Ca and Zn using atomicabsorption spectrophotometry (Varian Spectra 30,Varian Analytical Instruments, Sunnyvale, CA) afterfeed samples were dry-ashed at 450C. Total P wasdetermined by a colorimetric procedure described byAOAC ( 1984 ). Phytate content of the diets wasdetermined by the method of Harland and Oberleas(1986).Blood and Bone Measurements. Blood samples weretaken before the experimental diets were fed and atvarious intervals during the experiments. Collectionswere taken from the brachial region of each pig usingspecial-purpose (low Zn) trace element evacuatedtubes (Becton Dickinson Vacutainer System, Ruther-ford, NJ). Plasma was prepared for analysis bydiluting 1.0 mL of plasma with 4.0mL of deionizedwater for Zn determination by atomic absorptionspectrophotometry. In Exp. 1, blood samples weretaken from both fed pigs (wk 4, 0, and 12 ) and unfedpigs (initial and wk 6; feed was withheld for 14 hovernight). In Exp. 2, blood samples were taken fromfed pigs before the experimental diets were introducedand at wk 6 and 14 (w k 13 for two of the replicates).At the end of the experiments, all pigs were killedand the coccygeal vertebrae and 3rd and 4th metacar-pals were collected for Zn analysis. The feet and tailswere autoclaved at 120C for 20 min to facilitateremoval of muscle, skin, and connective tissue. Thebones were then dried at 105C overnight. The driedbones were then extrac ted with anhydrous ethyl etherfor 48 h (coccygeal vertebrae) o r 72 h (3rd and 4thmetacarpals). The dried, fat-free bones were thenashed at 700C for 12 h. The metacarpal bones wereground and subsampled, whereas the largest ver-tebrae ( unground ) were used for Zn determinations.

Bone samples were dissolved in 5 mL of 6 N HC1 anddiluted appropriately for Zn analysis via atomicabsorption spectrophotometry.Statistical Analysis. All ANOVA and regressionanalyses were performed using the GLM procedures ofSAS (1985) with a model appropriate fo r a ran-domized complete block design. Pen was the ex-perimental unit. In Exp. 1, a nonlinear regressionprocedure of SAS (1 98 5) was used t o determinebreakpoints using a model involving two linear splineswith no plateau (Robbins, 1986 ) wherein the depen-dent variable, bone or plasma Zn concentration, wasregressed on supplemental Zn intake, phytate:Zn, o r(Ca x phytate):Zn molar ratios. Treatment meanswere compared using orthogonal contrasts (i.e ., linear,quadratic, and cubic). Orthogonal polynomial esti-mates were generated using PROC IML@ (SAS,1985). In Exp. 2, Zn bioavailability was determinedusing multiple linear regression, slope-ratio analysis.The model included block and the amount (milli-grams/day) of Zn fed from each of the Zn sources (i.e.,two levels for ZnS04.H20 and one level for ZnO,ZnMET, and ZnLYS) such that four regression lineswith a common intercept were determined. Althoughthe experimental sources of Zn were added to thebasal diet at only a single level, the four replicatesrepresenting 0 mgkg of added Zn and the fourrepresenting either 7.5 o r 15 mgkg of added Zn gaveeight data points fo r the construction of a line of bestfit. Thus, slopes could be compared to the standardsource of Zn (ZnS04.HzO) that consisted of threelevels of Zn and 12 da ta points (Aoyagi et al., 1993).Shown in Figure 1 s a graphical representation of the

AA

91.BO

83.60

75.40

67.20 nt 8- .8 59.00 * I I0 0 10 20 30 400 Supplemental Zn i n take , mg/d

Figure 1.A graphical representation of the slope-ratioanalyses. The multiple regression equation was calcu-lated with block included in the model. Coccygealvertebrae Zn (micrograms of Zn/gram of dry, fat-freebone] was regressed on supplemental Zn supplied fromno supplemental Zn or diets supplemented withZnS04.HzO (ZnS041,A ; n methionine (ZnMET), ; Znlysine (ZnLYS), V; or ZnO, 0 .



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2684 WEDEKIND ET AL.Table 2. Effect of zinc level on pig performance (Exp. 1)

Supplemental Zn, m g k ePeriod Ob 5 10 20 40 80 SEM cvGrowing phase'ADFI, kg 1.84 1.93 1.89 1.81 1.80 1.82 .034 3.72ADG, kg .72 .7 8 .75 .72 .73 .73 ,009 2.45Gaidfeed .39 .40 .40 .40 .41 .40 .006 2.84

Finishing phasedADFI, kg 2.71 2.92 2.79 2.77 2.79 2.73 .062 4.45ADG, kg .89 .90 .87 .89 .88 .86 ,018 4.03Gaidfeed .33 .31 .31 .32 .32 .32 ,007 4.38

aSupplemental Zn was provided as ZnS04.HZO.bBasal diet supplied 32 mg of Znkg of diet in the growing diet and 27 mg of Zn/kg of diet in the finishing diet.'42-d period; average initia l weight was 24.5 kg.d43-d period for Replication 2, 57-d period for Replications 1 an d 3, and 64-d period fo r Replication 4. Average final weight was 104 kg.

data. The intercept was calculated by assuming thatblock effects summed to zero. The dependent varia-bles, metacarpal Zn concentration, coccygeal vertebraeZn concentration, and plasma Zn concentration, wereregressed on supplemental Zn intake. Differencesbetween slopes were tested using t-tests among allpairs only if F-tests fo r differences among slopes weresignificant ( P < .05).

ResultsPretest (D epletio n) Period. Approximately 5% of the

pigs developed signs of Zn deficiency (parakeratosis)by the end of the 5-wk pretest period. The frequency ofincidence was similar in the two experiments. In Exp.1, the average plasma Zn concentration at the end ofthe depletion period (unfed st at e) was .58 pg/mL. InExp. 2, the p lasma Zn concentration (fed s ta te ) was.29 pg/mL.Experiment 1 . Two animals were removed from thisexperiment because of listlessness and weight loss.These conditions did not seem to be associated with

dietary treatment. Zinc supplementa tion did not affect( P >. l o ) pig performance in either the growing phaseo r the finishing phase (Table 2) . Significant blockeffects (weight and sex) were noted during thegrowing and finishing phases ( P < .05). In general,barrows ate more feed and gained more weight thangilts and, as a consequence, deposited more Zn intissues. Gaidfeed was not affected by sex but wasgreater for the heavier replications (finishing phaseonly). Substantial increases in plasma Zn and bone Znoccurred as dietary Zn concentration increased (Tab le3 ) .Irrespective of the Zn intake, plasma Zn concentra-tions of unfed pigs were higher than those of fed pigs(Figure 2). This was especially true at the two lowestZn intakes, fo r which there was a twofold difference inplasma Zn concentrations between fed and unfed pigsand threefold differences in slope below the inflectionpoints. Zinc concentrations in plasma collected at wk10 and 12 (Table 3 ) responded linearly ( P < .05) toZn supplementation but were more variable (largerC V ) than previous blood collections.Zinc concentrations in bone (Table 3 ) increasedlinearly ( P < .001) at levels up to 20 mgkg

Table 3. Effect of dietary zinc on plasma and bone zinc concentrations (Exp. 1)Supplemental Zn, m@ga

Criterion Ob 5 10 20 40 80 SEhl cvPlasma Zn, pg/mLInitial (u nf ed ) ,550 .552 .596 .572 .593 .601 .025 8.64 wk (fe dJc d ,419 ,510 .910 .960 .889 1.008 .033 8.56 wk (u nfed )' ,926 .956 1.106 1.164 1.152 1.290 .053 9.6

10 wk (fed)' .606 .789 .814 .735 .982 1.092 ,060 14.312 wk (fed)' .657 .600 .671 .799 ,888 1.021 .061 15.9

Bone Zn, pglgeCoccygeal vertebraeCd 87.1 90.6 109.4 126.0 140.4 154.6 3.1 5.3MetacarpalsCd 127.0 126.8 157.2 194.5 203.3 237.8 4.9 5.6

aSupplemental Zn was provided as ZnSOq.HZ0.bBasal diet supplied 32 mgkg of Zn in the growing diet and 27 mgkg of Zn in the finishing diet.'Linear effect ( P < ,001).dQuadratic effect ( P< . O O l J .eZn concentration in bone expressed per g ram of dry, fat-free bone.by guest on February 25, 2013www.journalofanimalscience.orgDownloaded from


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ZINC BIOAVAILABILITY FOR SWINE 2685

50250

1.40 r

b, ,343 ,156-breakpoint 43.4, 188.2 55.3, 134.2.997- R2 ,996I I I I

40 80 120 160 200

10 0 4 wk , l e d

.20 breakpoint 18 .5 . .91 23.1, 1.12- R2 ,994 ,999I I I I

Figure 2. Regression of plasma Zn on supplementalZn intake from feed-grade ZnS04.HzO for plasmacollected at wk 4 (fed state, A) nd wk 6 (unfed state,+). The breakpoint was determined using a modelinvolving two linear splines with no plateau. The Y-intercept is represented by bo and the slope below thebreakpoint is represented by bl. Above the breakpoint,the slope of the line is represented by bz. Thebreakpoints, 18.5 and 23.1 mg/d of supplemental Zn,correspond to total dietary concentrations of 46 and 47mg/kg of Zn, respectively.

supplemental Zn. At supplemental Zn concentrations> 20 mgkg (43 mg/d of supplemental Zn intake;Figure 3 ) ) the slope was markedly less as Zn intakeincreased (quadratic effect; P < .001). The break-points determined in Exp. 1 for bone and plasma Znwere lower for gilts than for barrows but were notstatistically different ( P > . l o ) .Experiment 2. Addition of Zn did not affect ( P>. l o ) pig performance (Table 4). Significant blockeffects (weight and sex) were observed for animalperformance and tissue Zn concentrations ( P < . 0 5 ) .Bone and plasma Zn at wk 14 (Table 5 ) increasedlinearly ( P


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2686 WEDEKIND ET AL.Table 5. Effect of dietary zinc on plasma and bone zinc concentrations [Exp. 2)

Bone Zn, pglgbSuppl. Zn,malka

Znsource

Plasma Zn, pg/mLa CoccygealInitial 6 wk 14 wkC Metacarpals' vertebrae'

Od7.57.57.5

15

15

ZnSOq.H20ZnSOq.H20ZnS04.H20ZnMETZnLYSZnOPooled SEMcv

,271.263,312,300,298.302,014

9.5

.374,433,422,438.295,356,054

27.89

,527,640,687,612,600,677,019

6.12

65.5377.4586.1471.9269.8379.61

1.453.85

68.2478.1590.2176.6670.3282.942.185.61

aPlasma Zn values are from fed pigs.bZn concentration in bone expressed per gram of dry, fat-free bone.CLinear effect of ZnSOq.H20 ( P < ,051.dBasal diet supplied 32 mg/kg of Zn in the growing diet and 27 mgkg of Zn in the finishing diet.

index of Zn availability, however, data indicated thatZn from ZnLYS and ZnO, but not from ZnMET, waslower in bioavailability than Zn from ZnS04.H2O ( P . lo ) .

DiscussionThe current NRC Zn requirement of pigs (6 0 and

50 mgkg of Zn fo r the growing [20 to 50 kgl andfinishing [50 t o 110 kgl phases, respectively) wasbased on the level of Zn needed to maximize growth(Lewis et al., 1956, 1957; Smith et al., 19611, yet inour experiments the Zn concentration in a corn-soybean meal diet did not limit growth even though Zn

stores were depleted during the pretest period. Thus,if growth is the primary criterion for establishingnutrient requirements, then the current NRC require-ment for Zn is to o high. However, growth may not bethe best index of Zn status. Some minerals, such asZn, play important roles in immunocompetence andreproduction. The Zn concentration needed t o max-imize reproductive performance is higher than thatneeded t o maximize growth (Underwood, 1981) andmay also be higher for immune response whenanimals ar e stressed (Klasing, 1992). Unfortunately,there are currently no indices to delineate easily theZn requirement fo r maximizing reproductive or im-mune responses. Bone represents the major storagesite of Zn in the body. If bone Zn stores aremaximized, perhaps reproductive performance andimmune function would likewise be maximized. Thus,it is our opinion that the inflection point determined

Table 6. Relative bioavailability of zinc from various zinc sources (Exp. 2 )Dependent variable (Y) Regression equationa Zn source R B V ~ R2Metacarpal Zn, pglg" 65.9 ( ? 1.7) + ,6621 ( ? ,065) Xi ZnS04.HzO 100.0d .93

+ ,4001 ( ? ,129) Xz ZnMET 60.4e+ .4418 ( ? ,063) X , ZnO 66.7e+ ,2484 (f ,126) X3 ZnLYS 37.5f

Coccygeal vertebrae Zn, pg/gc 67.7 ( k 2.5) + ,7074 ( + ,097) Xi+ ,5968 ( ? ,190) Xz+ ,1721 ( + ,187) X3+ ,4917 ( ? ,094) X

ZnS04,HzO 100.0d .89ZnMET 84.4deZnLYS 24.3fZnO 69.5e

Plasma Zn, pg/mL (w k 14 ) .54 ( i 02) + ,0052 ( + ,00 1) Xi ZnS04.HzO 100.0d .82+ ,0050 (+ ,002) Xz ZnMET 95.4d+ ,0041 ( k ,002) X3 ZnLYS 78.7d+ ,0045 (+ ,001) 4 ZnO 87.0d

aMultiple linear regression of dependent variable ( Y ) on milligram dday of supplemental Zn intake (va lues in parentheses are s tandar dbRelative bioavailability es timate where ZnSO4.Hz0, the standard, is set at 100%.CZn concentration in bone expressed per gram of dry, fat -free bone.d,e,fMeans without the same superscript differ ( P < .05).

errors).



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ZINC BIOAVAILABILITY FOR SWINE 2687for bone and plasma Zn is an appropriate index of theZn requirement. Setting a requirement based onmaximization of tissue stores is not new. Some of therecommended dietary allowances (RDA, 1989) estab-lished for humans (i.e., vitamin C, vitamin BIZ, Ca,and Fe) are based on optimization of tissue stores.Data from Exp. 1 indicate a Zn requirement of 50 mg/kg of die t (i .e ., inflection points were 50, 45, 46, and47 mgk g of dietary Zn fo r coccygeal vertebrae,metacarpals, and plasma Zn [wk 4 and 61, respec-tively) for pigs in both the growing and finishingperiods.

Previous research by Wedekind e t al. (1992) withchicks demonstrated significant differences in Znbioavailability among ZnS04.H20, ZnO, and ZnMET.For example, when chicks were fed a corn-soybeanmeal diet, Zn from ZnMET was shown t o be 206%bioavailable relative to a ZnS04.H20 standard( l o o% ) ,whereas ZnO provided only 61%bioavailableZn. The present studies with pigs also showed thatZnO, relative t o ZnSOq-H20, provided only 68%bioavailable Zn (metacarpal Zn, 66.7%; coccygealvertebrae Zn, 69.5%). However, the finding thatZnMET did not provide more bioavailable Zn tha nZnS04.NaO is in contrast t o the chick data.

When ZnS04.H20 and ZnMET were compared inchicks fed a crystalline amino acid diet (Wedekind etal., 19921, the relative Zn bioavailability estimatesvaried greatly from estimates determined in soyisolate-dextrose and corn-soybean meal diets. In acrystalline amino acid diet, the Zn in ZnMET providedonly 17% more bioavailable Zn relative t o th eZnS04.H2O source. Thus, in a diet devoid of phytateand fiber, the ZnMET complex offered little advantageover inorganic Zn sources. These data suggest that thebioavailability of Zn in ZnMET, relative t o inorganicZn sources, may depend on the amounts of antagonis-tic factors (e.g., Ca, phytate, and fiber) that arepresent in the diet.

High dietary Ca accentuates the effect of phytate onZn bioavailability (Ober leas e t al., 1962; ODell et al.,1964; Ellis et al., 1982; Fordyce et al., 1987). Thediets used in the chick studies contained considerablymore Ca than the diets used in the current researchwith pigs (i.e., 1%Ca in corn-soybean meal chick dietsvs .64%Ca in the pig growing diets and .59%Ca in thepig finishing diets). It also has been established thatexcess dietary Ca increases the incidence of parakera-tosis in swine fed corn-soybean meal diets (Tuckerand Salmon, 1955; Lewis et al., 1956, 1957; Luecke etal., 1957). Thus, the lower Ca and phytate content ofthe swine diets may explain, in part, why ZnMET ( o rZnLYS) offered no advantage over inorganic Znsources in supplying bioavailable Zn in our swinediets, yet provided more Zn relative t o ZnS04.H20 inchick diets (Wedekind et al., 1992; diets that con-tained higher Ca levels). This hypothesis is partiallysupported by research conducted in chicks (Wedekind

et al., 1994) that indicated t ha t the bioavailability ofZn in ZnMET, relative t o ZnS04.H20, increased withincreasing Ca level. In addition, the phytate concen-tration in our most recent chick study was substan-tially higher than tha t in the present study (5 .9 vs 3.6mg of phyta te per g ram of diet, respectively). Thus,the discrepancies noted between poultry and swinestudies may be attributed more to differences in Caand phytate levels than to species differences. Ourfindings agree with those of Hill et al. (1986) andSwinkels et al. (19911, who also reported that in pigsthe bioavailability of Zn in Zn complexes and chelateswas no greater than that of Zn in Zn sulfate.

The overt symptoms of parakeratosis observed atthe end of the Zn-depletion period were not sustainedduring the growing and finishing periods. Signs ofparakeratosis disappeared within 1wk afte r pigs werefed the growing diets, even though some pigs exhibit-ing parakeratosis were allotted t o diets that were evenlower in Zn than the pretest diets. Apparently Zn wasnot as limiting during the growing and finishingperiods as during the starting phase, perhaps due toenvironmental contamination.

Plasma Zn concentrations increased over time frominitial to final determinations (Tables 3 and 5 ) , butvalues measured in fed pigs were generally lower thannormal ranges ( .70 t o 1.5 pg/mL; Puls, 1990).Comparisons were made between plasma Zn concen-trations in fed and unfed pigs in an attempt to reducethe variation seen in plasma Zn. Regardless ofwhether plasma was collected from fed o r unfed pigs,plasma Zn was more variable in assessing Zn statusthan was bone Zn, as evidenced by higher CV andSEM and lower R2. Plasma Zn was also less dis-criminating in assessing Zn bioavailability among Znsources. These findings are similar t o results obtainedwith chicks (Wedekind and Baker, 1990; Wedekind etal., 1992).

The phytate, Zn, and Ca contents of diets, expressedas molar ratios (phytate:Zn or [Ca x phytate1:Zn;molesk ilogram), have been used t o predict thebioavailability of Zn (Morris and Ellis, 1980; Fordyceet al., 1987). As shown in Figure 4a, there is abreakpoint in the response curve of plasma Zn at wk 4regressed on phytate:Zn indicating that a t a ratio of 6.5 tissue Zn storesmay become depleted. In Figure 4b are the results ofplotting bone Zn and wk 12 plasma Zn againstphytate:Zn. An inflection point occurred at a phytate:Zn molar ratio of 11.8. The lack of a growth responseto supplemental Zn is in good agreement with thefindings of Morris and Ellis (1980), who reported tha tin diets low in Ca ( .75%), growth of rats was notaffected by phytate:Zn molar ratios of 5 12. Thus, thegrowth ra te of pigs fed Zn-unsupplemented, corn-soybean meal basal diets in our study (phytate:Zn =12.3 for growing die t) would not have been expected t oincrease as a result of Zn addition.



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2688 WEDEKIND ET AL.1.2

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Figure 4. Regression of plasma Zn ( 0 )and bone Zn ( A ) on phytate:Zn or (Ca x phytate):Zn molar ratios. Thebreakpoint was determined using a model involving two linear splines with no plateau (a and c ) or a one-slopebroken line model (b and d ). (a) The regression of plasma Zn on the phytate:Zn molar ratio yielded the followingestimates: Y-intercept (bo) = .91, slope below the breakpoint (bl) = .015, slope above the breakpoint (bz) = .221,and the breakpoint = 6.5. (b ) The regression of bone Zn on the phytate:Zn molar ratio yielded the followingestimates: bo = 127.1, bl = 13.14, and breakpoint = 11.8. (c)The regression of plasma Zn on the (Ca x phytate):Znmolar ratio yielded the following estimates: bo = .91, bl = .108, b2 = 1.52, and the breakpoint = .94. (d) Theregression of bone Zn on the (Ca x phytate):Zn molar ratio yielded the following estimates: bo = 127.0, bl = 93.8,and the breakpoint = 1.65.

The response curves (Figures 4c and 4 d ) werevirtually identical when (Ca x phytate1:Zn molarratios were used instead of phytate:Zn; the break-points occurred at (Ca x phytate):Zn of .94 (w k 4plasma Zn) and 1.6 (bone Zn). The plot of bone Znagainst (Ca x phytate):Zn molar ratio is similar toth at observed for rats by Fordyce et al. (19871, exceptthat the inflection point in their study occurred at a(Ca x phytate):Zn molar ratio of 5.88, compared with1.65 in our study. In the study by Fordyce et al.(19871, the (Ca x phytate):Zn, but not the phytate:Zn, molar ratio was useful in predicting Zn availabil-ity. These discrepancies may be attributed to thenarrow range and low level of Ca and phytate

employed in our studies compared with those used inthe study by Fordyce et al. (1987). For example, the(Ca x phytate):Zn molar ratio ranged only from .45 o2.0 in our studies, compared with 0 to 9.5 in the studyof Fordyce et al. (1987) . Had our diets incorporated arange of levels of Ca a nd (o r) phytate, the results mayhave shown (Ca x phytate):Zn to be more predictiveof Zn utilization than phytate:Zn molar ratios.More information is needed to improve the u tility ofthese molar ratios in predicting Zn utilization. Ourdata and those of others (Davies et al., 1985; Fordyceet al., 1987) suggest tha t factors such as species, age,diet composition, diet processing, response criterion,and Zn source influence the predictive value of (Ca xphytate1:Zn molar ratios.



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ZINC BIOAVAILABILITY FOR SWINE 2689Implications

Although bone and plasma zinc concentrationincreased in response t o zinc supplementation, theresults of our studies suggest that for pigs fed corn-soybean meal diets zinc is not limiting for growth.Among the zinc sources tested in growing-finishingpigs, zinc sulfate provided more bioavailable zinc th anzinc oxide or zinc-lysine, and zinc bioavailability wasnumerically greater than for zinc-methionine. Con-trary to findings obtained in poultry studies, theorganic zinc sources did not provide more bioavailablezinc than zinc sulfate.

Literature CitedAOAC. 1984. Official Methods of Analysis ( 14 th Ed .) . Association of

Official Analytical Chemists, Arlington, VA.Aoyagi, S., D. H. Baker, and K. J. Wedekind. 1993. Estimates of

copper bioavailability from liver of different animal species andfrom feed ingredients derived from plants and animals. Poult.Sci. 72:1746.Davies, N. T., A.J.P. Carswell, and C. F. Mills. 1985. The effects ofvariation in d ietary calcium intake on the phytate-zinc interac-tion in rats. In: C. F. Mills, I. Bremmer, and J. K. Chesters(E d. ) Trace Elements in Man and Animals-TEMA 5. Aberdeen,U.K.

Ellis, R., E. R. Morris, and A. D. Hill. 1982. Bioavailability to rats ofiron an d zinc in calcium-iron-phytate an d calcium-zinc-phytatecomplexes. Nutr. Res. 2:319.

Fordyce, E. J., R. M. Forbes, K. R. Robbins, and J. W. Erdman, Jr .1987. Phytate x calciudzinc molar ratios: Are they predictiveof zinc bioavailability? J . Food Sci. 52:440.

Harland, B. F., and D. Oberleas. 1986. Anion exchange method fordetermination of phytate in foods: Collaborative study. J.Assoc.Off. Anal. Chem. 69:667.

Hill, D. A,, . R. Peo, Jr., A. J. Lewis, and J. D . Crenshaw. 1986.Zinc-amino acid complexes for swine. J. h i m . Sci. 63:121.Klasing, K. C. 1992. Nutrition an d immunity. What is known aboutfeeding animals for optimum immunocompetence? Large Anim.Vet. 47:16.

Lewis, P. K., Jr., W. G. Hoekstra, and R. H. Grummer. 1957.Restricted calcium feeding versus zinc supplementation for thecontrol of parakeratosis in swine. J. h i m . Sci. 16578.

Lewis, P. K., Jr., W. G. Hoekstra, R. H. Grummer, and R. H.Phillips. 1956. The effect of certain nutrit ional factors includingcalcium, phosphorus and zinc on parakeratosis in swine. J.Anim. Sci. 15:741.

Luecke, R. W., J. A. Hoefer, W. S. Brammell, and D. A. Schmidt.1957. Calcium and zinc in parakeratosis of swine. J. h i m . Sci.16:3.

Morris, E. R., and R. Ellis. 1980. Effect of dietary phytatehinc molarratio on growth and bone zinc response of rats fed semipurifieddiets. J . Nut r. 110:1037.

NRC. 1988. Nutrient Requirements of Swine (9th Ed.). NationalAcademy Press, Washington, DC.

Oberleas, D., M. E. Muhrer, and B. L.ODell. 1962. Effects of phyticacid on zinc availability and parakeratosis in swine. J. Anim.Sci. 21:57.

ODell, B. L., J. M. Yohe, and J. E. Savage. 1964. Zinc availability inthe chick as affected by phytate, calcium and ethylenedi-aminetetraacetate. Poult. Sci. 43:415.PUIS, R. 1990. Minera l Levels in Animal Health: Diagnostic Data .Clearbrook, Sherpa International, British Columbia, Canada.

RDA. 1989. Recommended Dietary Allowances (10th Ed.). NationalAcademy of Sciences, Washington, DC.

Robbins, K. R. 1986. A method, SAS program, and example forfitting the broken line to growth data. Univ. of Tennessee Res.Rep. 86-09. Univ. of Tennessee Agric. Exp. Sta., Knoxville.

SAS. 1985. SAS Users Guide: Sta tistics (Version 5 Ed .). SAS Inst .Inc., Cary, NC.

Smith, W. H., M . P. Plumlee, and W. M. Beeson. 1961. Zinc require-ment of the growing pig fed isolated soybean protein semi-purified rations. J. Anim. Sci. 20:128.

Swinkels, J.W.G.M., E. T. Kornegay, K. E. Webb, Jr., an d M. D.Lindemann. 1991. Comparison of inorganic and organic zincchelate in zinc depleted and repleted pigs. J. Anim. Sci.69(Suppl. 0 3 5 8 (Abstr.) .

Tucker, H. F., and W. D . Salmon. 1955. Parakeratosis or zincdeficiency disease in the pig. Proc. SOC. xp. Biol. Med. 88:613.

Underwood, E. J. 1981. The Mineral Nutrition of Livestock (2ndEd.). Commonwealth Agric. Bureaux, Slough, U.K.

Wedekind, K. J., an d D . H. Baker. 1990. Zinc bioavailability in feed-grade sources of zinc. J. Anim. Sci. 68:684.Wedekind, K., G. Collings, J. Hancock, and E. Titgemeyer. 1994. Thebioavailability of zinc-methionine relative t o zinc sulfate isaffected by calcium level. Poult. Sci. 73(Suppl. 1) :114 (Abs tr .) .

Wedekind, K. J. , A. E. Hortin, and D. H. Baker. 1992. Methodologyfor assessing zinc bioavailability: Eficacy estimates for zinc-methionine, zinc su lfate, an d zinc oxide. J. Anim. Sci. 70:178.



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