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Effect of Dietary Manganese and Age on Tissue Trace Mineral Composition of Broiler-Type Chicks as a Bioassay of Manganese Sources1,2
Effect of Dietary Manganese and Age on Tissue Trace Mineral Composition of Broiler-Type Chicks as a Bioassay of Manganese Sources1'2 J. R. BLACK,3 C. B. AMMERMAN4'5 P. R. HENRY, and R. D. MILES 6 University of Florida, Gainesville, Florida 32611 (Received for publication May 16, 1984)
1985 Poultry Science 64:688-693 INTRODUCTION Manganese is distributed widely throughout the body, although it is generally higher in bones (Underwood, 1977) and tissues rich in mitochondria (Maynard and Cotzias, 1955). Young animals have been shown to have higher intestinal absorption (70% compared with 2%) and tissue mineral concentrations than more mature animals (Cahill et al, 1980; Mena, 1981).
Black et al. (1984a,b) indicated that tissue uptake of manganese (Mn), resulting from high dietary concentrations, was a useful measure of relative bioavailability. Because absorption and tissue Mn concentration decrease the first few days of life, it would be of interest to determine if the sensitivity of this bioassay is influenced by age. The purpose of this experiment was to investigate the effects of age and dietary Mn concentration on tissue trace mineral composition in chicks to optimize a bioassay method. MATERIALS AND METHODS
1
Florida Agricultural Experiment Station Journal Series No. 5585. 2 The authors wish to acknowledge International Minerals & Chemical Corp., Mundelein, IL; Moorman Manufacturing Co., Quincy, IL, Occidental Chemical Co., Houston, TX; and Southeastern Minerals, Bainbridge, GA, for funds in support of this research and S. M. Free, Smith-Kline Animal Health Products, Philadelphia, PA, for assistance with the statistical analysis. 3 Central Soya Co., 1200 N. 2nd Street, Decatur, IN 46733. 4 To whom correspondence should be addressed. 5 Department of Animal Science. 6 Department of Poultry Science.
One hundred forty-four, day-old Cobb feather-sexed male chicks were used in a 4 x 3 factorial arrangement of treatments that included 0, 1000, 2000, or 3000 ppm Mn as reagent grade manganese sulfate monohydrate ( M n S 0 4 - H 2 0 ) added to a basal diet (Table 1) containing 112 ppm Mn (by analysis) for 1, 2, or 3 weeks. Two pen replicates of 6 chicks were assigned to each of 12 treatment combinations, weighed at Day 0, and fed the experimental diets in a wire floor, thermostatically controlled, electrically-heated Petersime battery.
688
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ABSTRACT An experiment was conducted with 144 Cobb broiler-type chicks in a 4 X 3 factorial arrangement of treatments to investigate the effects of dietary manganese (Mn) and age on trace mineral composition in tissues. Day-old chicks were fed a basal corn-soybean meal diet (112 ppm Mn) supplemented with 0, 1000, 2000, or 3000 ppm Mn as manganese sulfate monohydrate ( M n S 0 4 - H 2 0 ) for 1, 2, or 3 weeks. There were two pen replications per treatment combination and six chicks per pen. Liver, kidney, pancreas, skeletal muscle, and bone were excised from 4 chicks per replication. Plasma was pooled from the remaining 2 chicks, and all samples were frozen for subsequent mineral analysis. There was a reduction in average daily feed intake (P<.05) at the 3000 ppm dietary level accompanied by a nonsignificant decrease in average daily gain and an increase in feed per unit gain. Tissue Mn increased (P<.001) for all tissues as dietary Mn increased. Tissue Mn concentration was reduced with age in kidney, pancreas, muscle (P<.001), and plasma (P<.05). Manganese in bone was lowest (P<.01) at 1 week of age. Of those ages tested, the optimum for bioassay based on A criterion values appeared to be 3 weeks. The highest A criterion values were obtained at 3 weeks of age in kidney followed by bone at 2 weeks. High dietary Mn increased copper (Cu) concentration in liver (P<.05) and plasma (P<.05) and reduced iron (Fe) in pancreas (P<.001). Liver Fe was lower (P<.001) at 3000 ppm than at 1000 or 2000 ppm dietary Mn. Plasma zinc (Zn) was higher (P<.05) at all supplemental Mn levels than the unsupplemented control, and kidney Zn increased (P<.001) at 3000 ppm dietary Mn. (Key words: dietary manganese, age, tissue minerals, bioavailability)
MANGANESE BIOAVAILABILITY TABLE 1. Composition of basal diet Concentration
Item
(%)
2.50 1.70 1.00 .50 .40 .25 1.00 100.00
Chemical c o m p o s i t i o n 4 Manganese Iron Zinc Copper
(ppm) 112 580 58 12.5
1
55.65 37.00
As-fed basis.
by flameless AAS on a Perkin-Elmer 503 with HGA-2100 graphite furnace (Anonymous, 1974). Data were analyzed by two-way analysis of variance, and multiple linear regression was determined by the least squares method (Steel and Torrie, 1980). No significant interactions between dietary Mn and age were found; therefore, only main treatment effect means are presented. RESULTS
Feed intake decreased (P<.05) from 32.7 g/day in the unsupplemented control to 30.6 g/day at the 3000 ppm Mn (Table 2). This reduction was accompanied by a trend toward reduction in average daily gain (P = .067) from 22.2 to 19.8 g/day. Feed per unit gain was not different (P>.10), although it was numerically higher at 3000 ppm. Average daily feed intake and gain almost doubled between 1 and 3 weeks of age.
2
Ingredients supplied per kilogram of diet: vitamin A palmitate, 6400 IU; vitamin D 3 , 2200 ICU; menadione dimethylpyrimidinol bisulfite 2.2 mg; riboflavin, 4.4 mg; panthothenic acid, 13 mg; niacin, 40 mg; choline chloride, 500 mg; vitamin B I 2 , 22 ng; ethoxyquin, .0125%; manganese 60 mg; iron, 50 mg; copper, 6 mg; zinc, 36 mg. 'Manganese supplement added at the expense of equivalent weights of washed sand. 4
Dry matter basis, by analysis.
Chicks were allowed ad lib access to feed and tap water and were maintained on a 24-hr constant-light schedule. At 1, 2, or 3 weeks of age, all chicks from two pen replications of each dietary Mn treatment were individually weighed, and feed consumption was determined per pen. Four chicks from each pen were randomly selected for tissue analysis, and blood was drawn by anterior heart puncture from the remaining two chicks. Blood was pooled, and plasma was saved for mineral analysis. Chicks were killed by cervical dislocation, and liver, kidney, pancreas, pectoralis major muscle, and right tibia were excised and frozen in heat-sealed polyethylene bags for subsequent mineral analysis. Feed and tissue Mn, zinc (Zn), iron (Fe), and copper (Cu) were determined by flame atomic absorption spectrophotometry (AAS) on a Perkin-Elmer Model 5000 (Anonymous, 1982), except for plasma Mn, which was determined
TABLE 2. Effect of dietary manganese (Mn) and age on feed intake, gain, and feed conversion in chicks Treatment Added Mn
Age
(ppm)1
(weeks)
0 1000 2000 3000 SE3
Avg initial weight
Avg daily intake
Avg daily
gain
Feed/ unit gain
(g) Mn level effects 2 51.0 50.9 51.0 51.0 1.9
32.7 32.6 32.9 30.6 .56
22.2 22.0 22.1 19.8 .66
1.49 1.48 1.49 1.56 .04
14.8 20.9 28.8 .57
1.52 1.47 1.52 .04
Age effects" 1 2 3 SE
50.7 51.2 51.0 1.7
22.3 30.7 43.7 .49
Statistical significance Mn Age
NS5 NS
<.05 <.001
NS NS < . 0 0 1 NS
•Basal diet contained 112 ppm Mn. Supplemental Mn as reagent grade MnS0 4 *H 2 0. 2 Each value represents the mean of six pens, 6 chicks per pen. 3
Pooled standard error.
"Each value represents the mean of eight pens, 6 chicks per pen. 5
Nonsignificant (P>.05).
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Ingredients 1 Corn, ground yellow Soybean meal, dehulled (48.5% crude protein) Corn oil Dicalcium p h o s p h a t e ( 1 8 . 5 % P, 22% Ca) Limestone, ground Microingredients 2 Salt, iodized DL-Methionine Filler, washed sand 3 Total
689
690
BLACK ET AL. TABLE 4. Effect of age on tissue trace mineral composition in chicks1 Mineral concentration, ppm, dry matter basis Manganese
Iron
1 2 3 SE 3
14.4 15.0 15.2 .45
Liver 200' 22.1' 163 21.1 238 15.7 11 .7
1 2 3 SE
25.5' 23.0 19.9 .84
283 269 288 9
Age
Copper
Zinc
(weeks) 74 78 76 2.5
Kidney TABLE 3. Effect of dietary manganese (Mn) on tissue trace mineral composition in chicks1
Added Mn
Mineral concentration, ppm, dry matter basis Manganese
Iron
0 1000 2000 3000 SE5
9.6* 15.0 17.1 17.8 .51
1723 227 230 171 13
0 1000 2000 3000 SE
3
Copper
Zinc
13.5' 10.5 9.6 .69
1 2 3 SE
274' 204 163 7
Liver
71.8' 55.3 40.8 3.0
1175 98 94 6.0
Muscle 18.7 4 18.8 19.1 22.0
71 78 76 80 2.9
Kidney 283 276 280 280 10
654 70 71 1.3
Pancreas
(ppm)'
10.0 20.0 27.8 33.5 .97
25.8' 21.2 17.5 1.1
21.9 21.4 20.3 22.5 1.3
69
3
63
69 72 1.5
1 2 3 SE
1.90' 1.22 .84 .08
63' 42 33 1.9
19.3' 8.4 5.8 .7
26' 22 20 .5
'Each value represents the main treatment effect mean of 32 chicks. 'Effect of age (P<.001). 3
Pooled standard error.
"Effect of age (P<.01). 5
Effect of age (P<.05).
Pancreas 0 1000 2000 3000 SE
5.23 9.3 15.1 15.3 .80
0 1000 2000 3000 SE
1.063 1.13 1.22 1.86 .10
250 3 231 189 185
57.4 57.1 50.8 58.5 3.5
98 102 105 106 6.9
Muscle 45 45 49 45 2.1
12.5 10.5 11.6 10.1
23 22 23 22
1 Each value represents the main treatment effect mean of 24 chicks.
'Basal diet contained 112 ppm Mn, supplemental Mn as reagent grade MnS0 4 - H 2 0 . 3
Effect of dietary Mn (P<.001).
"Effect of dietary Mn (P<.05). 5
Pooled standard error.
Kidney Mn increased (P<.001) from 10.0 to 33.5 ppm as supplemental Mn increased from 0 to 3000 ppm (Table 3) and decreased (P<.001) with age (Table 4). Kidney Cu decreased (P<.001) with age (Table 4) but not with Mn treatment. Kidney Zn increased (P<.01) with age and with Mn supplementation (P<.001). Multiple linear regression analysis yielded an equation with a correlation coefficient of r = .98 indicating that 96% of the variance in kidney Mn could be accounted for by the regression model (Table 5). Manganese in pancreas increased (P<.001) from 5.2 ppm in the control group to 15.3 ppm in the 3000 ppm treatment group (Table 3) but decreased (P<.001) with age from 13.5 to 9.6 ppm. Pancreas Fe (Table 3) was reduced
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Liver Mn increased (P<.001) from 9.6 to 17.8 ppm as dietary Mn increased (Table 3) but did not change with increasing age (Table 4). Copper in liver increased (P<.05) with increasing dietary Mn and decreased (P<.001) with advancing age. Iron in liver was lower (P<.001) at 3000 ppm Mn than at 1000 or 2000 ppm, but it was not different from the unsupplemented controls and was lowest (P<.001) at 2 weeks of age. Multiple linear regression of age and liver Mn vs. dietary Mn yielded an equation with a correlation coefficient r = .915 (Table 5).
MANGANESE BIOAVAILABILITY TABLE 5. Multiple linear regression analysis of tissue manganese (Mn) with respect to dietary Mn and age
Regression equation
Liver1 Kidney 1 Pancreas1 Muscle1 Bone 1 Plasma2
t = 10.06 t = 16.74 t = 9.69 t = 2.00 t=-6.52 t = 6.87
+ .0027x + .0078x + .0036x + .O0O25x + .0522x + .0025x
+ .403y -2.824y -1.936y + 7.096y + 7.096y -1.556y
.915 .980 .931 .945 .987 .969
! Where t equals tissue Mn (ppm); x equals dietary Mn (ppm); and y equals age (wk). Regression equation represents 96 chicks. 2 Where t equals plasma Mn (jug/dl); x equals dietary Mn (ppm); and y equals age (wk). Regression equation represents 24 pens pooled from 2 chicks per pen.
(P<.001) by dietary Mn. Pancreas Zn (P<.05), Fe, and Cu (P<.001) were reduced with increasing age. The multiple linear regression model (Table 5) fit the data (r = .931) but not as closely as for kidney. Muscle Mn increased (P<.001) from 1.06 to 1.86 ppm as dietary Mn increased. The greatest increase in muscle Mn occurred between 2000 and 3000 ppm dietary Mn (Table 3). All minerals measured in muscle (Table 4) decreased (P<.001) with age. The multiple linear regression model provided a better fit of the muscle data (r = .945) than for pancreas or liver. Bone ash as a percentage of dry, fat-free bone increased (P<.001) from 40.2 to 41.7 and 44.4% (SE = .48) with age but was not affected by dietary Mn level. Manganese in bone ash increased (P<.001) linearly from 13.0 to 52.5, 110.6, and 167.7 ppm, ash weight basis (SE = 4.5 3), as dietary Mn increased. In contrast to soft tissue, Mn in bone was increased (P<.01) by age from 75.2 to 93.3 and 89.4 ppm, ash weight basis (SE = 3.92). The regression model for bone Mn (Table 5) provided the best fit of all tissues (r = .987). Plasma Mn increased (P<.001) from 4.18 to 11.95 Mg/dl (Table 6) and plasma Cu increased (P<.05) from 16.0 to 25.7 jug/dl as dietary Mn increased. Plasma Zn was higher (P<.05) for all Mn-supplemented treatments than controls. Manganese (P<.05) and Zn (P<.01) decreased
with age. Multiple linear regression (Table 5) indicated linear effects of dietary Mn level and age on plasma Mn (r = .969). DISCUSSION
Manganese concentration in all tissues was increased linearly with increasing Mn in diet, which is in agreement with previous studies (Southern and Baker, 1983; Black et ah, 1984a,b). Polynomial regression of liver Mn uptake with respect to dietary Mn level indicated a significant quadratic effect (r = .973) in the range measured. This may be related to absorption changes, which occur in response to dietary manganese level (Suso and Edwards, 1969). The observed reduction in liver accumulation is accompanied by an increased rate of accumulation in muscle between 20G0 and 3000 ppm dietary Mn. This supports other studies that indicated that the capacity of liver
TABLE 6. Effect of dietary manganese (Mn) and age on the mineral composition of plasma in chicks Mairl treatment effects
Treaa • n e n t Added Mn
Age
(ppm) 1
(weeks)
Mineral concentratic in (Mg/dl) Mn Zinc Copper
Manganese level effects2 0
1000 2000 3000 SE3
4.18 6.07 7.96 11.95
140 184 182 182
16.0 21.3 24.7 25.7
.70
10
2.2
Age effects" 1 2 3 SE3
9.08 7.58 5.97 .60
194 174 148 9
24.3 19.4 22.1 1.9
Statistical significance Mn Age
<.001 <.05
<.05 <.01
<.05 NSS
1 Basal diet contained 112 ppm. Supplemental Mn as reagent grade MnSO„ - H 2 0 . 2 Each value represents the mean of six pens, pooled from 2 chicks per pen.
'Pooled standard error. "Each value represents the mean of eight pens, pooled from 2 chicks per pen. 5
Nonsignificant (P>.05).
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Tissue
Correlation coefficient
691
692
BLACK ET AL. TABLE 7. Coefficient of variation and lambda criterion values of chick tissues as related to age Tissue
Age
Liver
Kidney
Pancreas
Muscle
Bone
Plasma
76.3 73.2 71.8
36.5 52.2 46.2
(weeks) Coefficient of variation1 1 2 3
32.1 26.0 24.4
39.6 45.7 46.0
49.0 52.9 50.4
35.3 41.0 57.1
1 2 3
6.95 6.87 5.75
7.72 7.41 8.49
6.00 5.72 7.56
4.18 5.00 4.63
7.91 8.42 8.36
1
Coefficient of variation = (SD/mean) X 100.
2
Lambda criterion values = slope/SD. For convenience, values have been multiplied by 10 4 .
to excrete Mn can become overloaded, leading to accumulation in other tissues (Hall et al, 1981). Manganese concentrations in kidney, pancreas, muscle, and plasma were reduced with age, whereas that in bone was increased and that in liver was not affected. Such reductions in soft tissue have been associated with dilution by growth of the organs (Rehnberg et al, 1981), changes in blood flow to the organ and permeability of cellular membranes (Task Group on Metal Accumulation, 1973), reduced intestinal absorption of particulate matter (Le Feure and Joel, 1977), development of excretory mechanism (Miller et al, 1975), and reduced tissue retention (Mena, 1981). Level of dietary Mn influenced tissue concentration of several trace minerals. The mutual antagonism of Mn and Fe has been documented (Diez-Ewald et al, 1968). Liver and pancreas Fe were reduced by dietary Mn. Plasma and kidney Zn were increased by dietary Mn. Ivan and Grieve (1975) noted an increased liver, kidney and heart Zn in Mn-supplemented calves. In contrast, Hamilton et al. (1979) found supplemental Zn reduced Mn in the liver of Japanese quail. Dietary Mn increased Cu in the present study in liver and plasma. This agrees with other reports of increased liver (Anke et al, 1973) and plasma Cu (Gubler et al., 1954) in Mn-supplemented goats and rats, respectively. It has been demonstrated (Black et al, 1984a,b) that tissue accumulation of Mn at high dietary intakes is a sensitive bioassay for
7.55 7.28 7.89
Mn in chicks. Because there is a difference in absorption and tissue level with age, it was of interest to determine the age that provided the most sensitive bioassay. To evaluate age differen ces, coefficient of variation [CV = (SLV mean) X 100] and X criterion values (X = slope/SD) were determined (Table 7) for various tissues at 1, 2, and 3 weeks of age. In kidney and muscle, CV increased with age, while in bone and liver it decreased with no pattern observed in pancreas or plasma. The X values were higher for all tissues except liver at 3 weeks compared to 1 week. It had been determined previously (Black et al., 1984a) that bone and kidney provided the most sensitive bioassay for Mn. The highest X values were obtained with kidney at 3 weeks followed by bone at 2 weeks. These data suggest, based on X criterion values, that a 3-week bioassay study may provide better differentiation among sources than a shorter trial. REFERENCES Anke, M., A. Hennig, B. Groppel, G. Dittrich, and M. Grun, 1973. Manganmangel beim Wiederkauer. 4. Der Einfluss des Manganmangels auf den Gehalt neugeborener Lammer an Fett, Protein, Mangan, Asche, Kalzium, Phosphor, Zink and Kupfer. Arch. Tierernaehr. 23:213-223. Anonymous, 1974. Analytical Methods for Atomic Absorption Spectroscopy Using the HGA Graphite Furnace. The Perkin-Elmer Corp., Norwalk, CT. Anonymous, 1982. Analytical Methods for Atomic Absorption Spectrophotometry. The PerkinElmer Corp., Norwalk, CT. Black, J. R., C. B. Ammerman, P. R. Henry, and R. D. Miles, 1984a. Tissue manganese uptake as a
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Lambda criterion values2
MANGANESE BIOAVAILABILITY
1403-1408. Maynard, L. S., and G. C. Cotzias, 1955. The partition of manganese among organs and intracellular organelles of the rat. J. Biol. Chem. 2 1 4 : 4 8 9 495. Mena, I., 1981, Manganese. Pages 2 3 3 - 2 7 0 in Disorders of Mineral Metabolism. Vol. I. F. Bronner and J. W. Coburn, ed. Academic Press, New York, NY. Miller, S. T., G. C. Cotzias, and H. A. Evert, 1975. Control of tissue manganese: Initial absence and sudden emergence of excretion in the neonatal mouse. Am. J. Physiol. 229:1080-1084. Rehnberg, G. L., J. F. Hein, S. D. Carter, R. S. Linko, and J. W. Laskey, 1981. Chronic ingestion of M n 3 0 4 by young rats: tissue accumulation, distribution, and depletion. J. Toxicol. Environ. Health 7:263-272. Southern, L. L., and D. H. Baker, 1983. Excess manganese ingestion in the chick. Poultry Sci. 62:642-646. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics: A Biometrical Approach. 2nd ed. McGraw-Hill Book Co., New York, NY. Suso, F. A., and H. M. Edwards, Jr, 1969. Whole body counter studies on the absoprtion of 6 0 Co, 59 Fe, 54 Mn, and 6 s Zn by chicks, as affected by their dietary levels and other supplemental divalent elements. Poultry Sci. 48:933-938. Task Group on Metal Accumulation, 1973. Accumulation of toxic metals with special reference to their absorption, excretion and biological halftimes. Environ. Physiol. Biochem. 3:65—107. Underwood, E. J., 1977. Trace Elements in Human and Animal Nutrition. 4th ed. Academic Press, New York, NY.
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measure-of manganese bioavailability. Nutr. Rep. Int. 29:807-814. Black, J. R., C. B. Ammerman, P. R. Henry, and R. D. Miles, 1984b. Biological availability of manganese sources and effects of high dietary manganese on tissue mineral composition of broiler-type chicks. Poultry Sci. 63:1999-2006. Cahill, D. F., M. S. Bercegeay, R. C. Haggerty, J. E. Gerding, and L. E. Gray, 1980. Age-related retention and distribution of ingested M n 3 0 4 in the rat. Toxico. Appl. Pharmacol. 5 3 : 8 3 - 9 1 . Diez-Ewald, M., L. R. Weintraub, and W. H. Crosby, 1968. Interrelationshp of iron and manganese metabolism. Proc. Soc. Exp. Biol. Med. 129: 448-455. Gubler, C. J., D. S. Taylor, E. J. Eichwald, G. E. Cartwright, and M. M. Wintrobe, 1954. Copper metabolism. XII. Influence of manganese on metabolism of copper. Proc. Soc. Exp. Biol. Med. 86:223-227. Hall, E. D., H. W. Symonds, and W. M. Allen, 1981. The roles of the liver and gut in Mn homeostasis in cattle. Pages 89—91 in Trace Element Metabolism in Man and Animals — IV. J. McC. Howell, J. M. Gawthorne and C. L. White, ed. Aust. Acad. Sci., Canberra. Hamilton, R. P., M. R. S. Fox, B. E. Fry, Jr., A.O.L. Jones, and R. M. Jacobs, 1979. Zinc interference with copper, iron and manganese in young Japanese quail. J. Food Sci. 44:738—741. Ivan, M., and C. M. Grieve, 1975. Effects of zinc, copper, and manganese supplementation of high-concentrate ration on digestibility, growth, and tissue content of Holstein calves. J. Dairy Sci. 58:410-415. Le Feure, M. E., and D. D. Joel, 1977. Intestinal absorption of particulate matter. Life Sci. 21:
693
1985 Poultry Science 64:688-693 INTRODUCTION Manganese is distributed widely throughout the body, although it is generally higher in bones (Underwood, 1977) and tissues rich in mitochondria (Maynard and Cotzias, 1955). Young animals have been shown to have higher intestinal absorption (70% compared with 2%) and tissue mineral concentrations than more mature animals (Cahill et al, 1980; Mena, 1981).
Black et al. (1984a,b) indicated that tissue uptake of manganese (Mn), resulting from high dietary concentrations, was a useful measure of relative bioavailability. Because absorption and tissue Mn concentration decrease the first few days of life, it would be of interest to determine if the sensitivity of this bioassay is influenced by age. The purpose of this experiment was to investigate the effects of age and dietary Mn concentration on tissue trace mineral composition in chicks to optimize a bioassay method. MATERIALS AND METHODS
1
Florida Agricultural Experiment Station Journal Series No. 5585. 2 The authors wish to acknowledge International Minerals & Chemical Corp., Mundelein, IL; Moorman Manufacturing Co., Quincy, IL, Occidental Chemical Co., Houston, TX; and Southeastern Minerals, Bainbridge, GA, for funds in support of this research and S. M. Free, Smith-Kline Animal Health Products, Philadelphia, PA, for assistance with the statistical analysis. 3 Central Soya Co., 1200 N. 2nd Street, Decatur, IN 46733. 4 To whom correspondence should be addressed. 5 Department of Animal Science. 6 Department of Poultry Science.
One hundred forty-four, day-old Cobb feather-sexed male chicks were used in a 4 x 3 factorial arrangement of treatments that included 0, 1000, 2000, or 3000 ppm Mn as reagent grade manganese sulfate monohydrate ( M n S 0 4 - H 2 0 ) added to a basal diet (Table 1) containing 112 ppm Mn (by analysis) for 1, 2, or 3 weeks. Two pen replicates of 6 chicks were assigned to each of 12 treatment combinations, weighed at Day 0, and fed the experimental diets in a wire floor, thermostatically controlled, electrically-heated Petersime battery.
688
Downloaded from http://ps.oxfordjournals.org/ at East Tennessee State University on June 25, 2015
ABSTRACT An experiment was conducted with 144 Cobb broiler-type chicks in a 4 X 3 factorial arrangement of treatments to investigate the effects of dietary manganese (Mn) and age on trace mineral composition in tissues. Day-old chicks were fed a basal corn-soybean meal diet (112 ppm Mn) supplemented with 0, 1000, 2000, or 3000 ppm Mn as manganese sulfate monohydrate ( M n S 0 4 - H 2 0 ) for 1, 2, or 3 weeks. There were two pen replications per treatment combination and six chicks per pen. Liver, kidney, pancreas, skeletal muscle, and bone were excised from 4 chicks per replication. Plasma was pooled from the remaining 2 chicks, and all samples were frozen for subsequent mineral analysis. There was a reduction in average daily feed intake (P<.05) at the 3000 ppm dietary level accompanied by a nonsignificant decrease in average daily gain and an increase in feed per unit gain. Tissue Mn increased (P<.001) for all tissues as dietary Mn increased. Tissue Mn concentration was reduced with age in kidney, pancreas, muscle (P<.001), and plasma (P<.05). Manganese in bone was lowest (P<.01) at 1 week of age. Of those ages tested, the optimum for bioassay based on A criterion values appeared to be 3 weeks. The highest A criterion values were obtained at 3 weeks of age in kidney followed by bone at 2 weeks. High dietary Mn increased copper (Cu) concentration in liver (P<.05) and plasma (P<.05) and reduced iron (Fe) in pancreas (P<.001). Liver Fe was lower (P<.001) at 3000 ppm than at 1000 or 2000 ppm dietary Mn. Plasma zinc (Zn) was higher (P<.05) at all supplemental Mn levels than the unsupplemented control, and kidney Zn increased (P<.001) at 3000 ppm dietary Mn. (Key words: dietary manganese, age, tissue minerals, bioavailability)
MANGANESE BIOAVAILABILITY TABLE 1. Composition of basal diet Concentration
Item
(%)
2.50 1.70 1.00 .50 .40 .25 1.00 100.00
Chemical c o m p o s i t i o n 4 Manganese Iron Zinc Copper
(ppm) 112 580 58 12.5
1
55.65 37.00
As-fed basis.
by flameless AAS on a Perkin-Elmer 503 with HGA-2100 graphite furnace (Anonymous, 1974). Data were analyzed by two-way analysis of variance, and multiple linear regression was determined by the least squares method (Steel and Torrie, 1980). No significant interactions between dietary Mn and age were found; therefore, only main treatment effect means are presented. RESULTS
Feed intake decreased (P<.05) from 32.7 g/day in the unsupplemented control to 30.6 g/day at the 3000 ppm Mn (Table 2). This reduction was accompanied by a trend toward reduction in average daily gain (P = .067) from 22.2 to 19.8 g/day. Feed per unit gain was not different (P>.10), although it was numerically higher at 3000 ppm. Average daily feed intake and gain almost doubled between 1 and 3 weeks of age.
2
Ingredients supplied per kilogram of diet: vitamin A palmitate, 6400 IU; vitamin D 3 , 2200 ICU; menadione dimethylpyrimidinol bisulfite 2.2 mg; riboflavin, 4.4 mg; panthothenic acid, 13 mg; niacin, 40 mg; choline chloride, 500 mg; vitamin B I 2 , 22 ng; ethoxyquin, .0125%; manganese 60 mg; iron, 50 mg; copper, 6 mg; zinc, 36 mg. 'Manganese supplement added at the expense of equivalent weights of washed sand. 4
Dry matter basis, by analysis.
Chicks were allowed ad lib access to feed and tap water and were maintained on a 24-hr constant-light schedule. At 1, 2, or 3 weeks of age, all chicks from two pen replications of each dietary Mn treatment were individually weighed, and feed consumption was determined per pen. Four chicks from each pen were randomly selected for tissue analysis, and blood was drawn by anterior heart puncture from the remaining two chicks. Blood was pooled, and plasma was saved for mineral analysis. Chicks were killed by cervical dislocation, and liver, kidney, pancreas, pectoralis major muscle, and right tibia were excised and frozen in heat-sealed polyethylene bags for subsequent mineral analysis. Feed and tissue Mn, zinc (Zn), iron (Fe), and copper (Cu) were determined by flame atomic absorption spectrophotometry (AAS) on a Perkin-Elmer Model 5000 (Anonymous, 1982), except for plasma Mn, which was determined
TABLE 2. Effect of dietary manganese (Mn) and age on feed intake, gain, and feed conversion in chicks Treatment Added Mn
Age
(ppm)1
(weeks)
0 1000 2000 3000 SE3
Avg initial weight
Avg daily intake
Avg daily
gain
Feed/ unit gain
(g) Mn level effects 2 51.0 50.9 51.0 51.0 1.9
32.7 32.6 32.9 30.6 .56
22.2 22.0 22.1 19.8 .66
1.49 1.48 1.49 1.56 .04
14.8 20.9 28.8 .57
1.52 1.47 1.52 .04
Age effects" 1 2 3 SE
50.7 51.2 51.0 1.7
22.3 30.7 43.7 .49
Statistical significance Mn Age
NS5 NS
<.05 <.001
NS NS < . 0 0 1 NS
•Basal diet contained 112 ppm Mn. Supplemental Mn as reagent grade MnS0 4 *H 2 0. 2 Each value represents the mean of six pens, 6 chicks per pen. 3
Pooled standard error.
"Each value represents the mean of eight pens, 6 chicks per pen. 5
Nonsignificant (P>.05).
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Ingredients 1 Corn, ground yellow Soybean meal, dehulled (48.5% crude protein) Corn oil Dicalcium p h o s p h a t e ( 1 8 . 5 % P, 22% Ca) Limestone, ground Microingredients 2 Salt, iodized DL-Methionine Filler, washed sand 3 Total
689
690
BLACK ET AL. TABLE 4. Effect of age on tissue trace mineral composition in chicks1 Mineral concentration, ppm, dry matter basis Manganese
Iron
1 2 3 SE 3
14.4 15.0 15.2 .45
Liver 200' 22.1' 163 21.1 238 15.7 11 .7
1 2 3 SE
25.5' 23.0 19.9 .84
283 269 288 9
Age
Copper
Zinc
(weeks) 74 78 76 2.5
Kidney TABLE 3. Effect of dietary manganese (Mn) on tissue trace mineral composition in chicks1
Added Mn
Mineral concentration, ppm, dry matter basis Manganese
Iron
0 1000 2000 3000 SE5
9.6* 15.0 17.1 17.8 .51
1723 227 230 171 13
0 1000 2000 3000 SE
3
Copper
Zinc
13.5' 10.5 9.6 .69
1 2 3 SE
274' 204 163 7
Liver
71.8' 55.3 40.8 3.0
1175 98 94 6.0
Muscle 18.7 4 18.8 19.1 22.0
71 78 76 80 2.9
Kidney 283 276 280 280 10
654 70 71 1.3
Pancreas
(ppm)'
10.0 20.0 27.8 33.5 .97
25.8' 21.2 17.5 1.1
21.9 21.4 20.3 22.5 1.3
69
3
63
69 72 1.5
1 2 3 SE
1.90' 1.22 .84 .08
63' 42 33 1.9
19.3' 8.4 5.8 .7
26' 22 20 .5
'Each value represents the main treatment effect mean of 32 chicks. 'Effect of age (P<.001). 3
Pooled standard error.
"Effect of age (P<.01). 5
Effect of age (P<.05).
Pancreas 0 1000 2000 3000 SE
5.23 9.3 15.1 15.3 .80
0 1000 2000 3000 SE
1.063 1.13 1.22 1.86 .10
250 3 231 189 185
57.4 57.1 50.8 58.5 3.5
98 102 105 106 6.9
Muscle 45 45 49 45 2.1
12.5 10.5 11.6 10.1
23 22 23 22
1 Each value represents the main treatment effect mean of 24 chicks.
'Basal diet contained 112 ppm Mn, supplemental Mn as reagent grade MnS0 4 - H 2 0 . 3
Effect of dietary Mn (P<.001).
"Effect of dietary Mn (P<.05). 5
Pooled standard error.
Kidney Mn increased (P<.001) from 10.0 to 33.5 ppm as supplemental Mn increased from 0 to 3000 ppm (Table 3) and decreased (P<.001) with age (Table 4). Kidney Cu decreased (P<.001) with age (Table 4) but not with Mn treatment. Kidney Zn increased (P<.01) with age and with Mn supplementation (P<.001). Multiple linear regression analysis yielded an equation with a correlation coefficient of r = .98 indicating that 96% of the variance in kidney Mn could be accounted for by the regression model (Table 5). Manganese in pancreas increased (P<.001) from 5.2 ppm in the control group to 15.3 ppm in the 3000 ppm treatment group (Table 3) but decreased (P<.001) with age from 13.5 to 9.6 ppm. Pancreas Fe (Table 3) was reduced
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Liver Mn increased (P<.001) from 9.6 to 17.8 ppm as dietary Mn increased (Table 3) but did not change with increasing age (Table 4). Copper in liver increased (P<.05) with increasing dietary Mn and decreased (P<.001) with advancing age. Iron in liver was lower (P<.001) at 3000 ppm Mn than at 1000 or 2000 ppm, but it was not different from the unsupplemented controls and was lowest (P<.001) at 2 weeks of age. Multiple linear regression of age and liver Mn vs. dietary Mn yielded an equation with a correlation coefficient r = .915 (Table 5).
MANGANESE BIOAVAILABILITY TABLE 5. Multiple linear regression analysis of tissue manganese (Mn) with respect to dietary Mn and age
Regression equation
Liver1 Kidney 1 Pancreas1 Muscle1 Bone 1 Plasma2
t = 10.06 t = 16.74 t = 9.69 t = 2.00 t=-6.52 t = 6.87
+ .0027x + .0078x + .0036x + .O0O25x + .0522x + .0025x
+ .403y -2.824y -1.936y + 7.096y + 7.096y -1.556y
.915 .980 .931 .945 .987 .969
! Where t equals tissue Mn (ppm); x equals dietary Mn (ppm); and y equals age (wk). Regression equation represents 96 chicks. 2 Where t equals plasma Mn (jug/dl); x equals dietary Mn (ppm); and y equals age (wk). Regression equation represents 24 pens pooled from 2 chicks per pen.
(P<.001) by dietary Mn. Pancreas Zn (P<.05), Fe, and Cu (P<.001) were reduced with increasing age. The multiple linear regression model (Table 5) fit the data (r = .931) but not as closely as for kidney. Muscle Mn increased (P<.001) from 1.06 to 1.86 ppm as dietary Mn increased. The greatest increase in muscle Mn occurred between 2000 and 3000 ppm dietary Mn (Table 3). All minerals measured in muscle (Table 4) decreased (P<.001) with age. The multiple linear regression model provided a better fit of the muscle data (r = .945) than for pancreas or liver. Bone ash as a percentage of dry, fat-free bone increased (P<.001) from 40.2 to 41.7 and 44.4% (SE = .48) with age but was not affected by dietary Mn level. Manganese in bone ash increased (P<.001) linearly from 13.0 to 52.5, 110.6, and 167.7 ppm, ash weight basis (SE = 4.5 3), as dietary Mn increased. In contrast to soft tissue, Mn in bone was increased (P<.01) by age from 75.2 to 93.3 and 89.4 ppm, ash weight basis (SE = 3.92). The regression model for bone Mn (Table 5) provided the best fit of all tissues (r = .987). Plasma Mn increased (P<.001) from 4.18 to 11.95 Mg/dl (Table 6) and plasma Cu increased (P<.05) from 16.0 to 25.7 jug/dl as dietary Mn increased. Plasma Zn was higher (P<.05) for all Mn-supplemented treatments than controls. Manganese (P<.05) and Zn (P<.01) decreased
with age. Multiple linear regression (Table 5) indicated linear effects of dietary Mn level and age on plasma Mn (r = .969). DISCUSSION
Manganese concentration in all tissues was increased linearly with increasing Mn in diet, which is in agreement with previous studies (Southern and Baker, 1983; Black et ah, 1984a,b). Polynomial regression of liver Mn uptake with respect to dietary Mn level indicated a significant quadratic effect (r = .973) in the range measured. This may be related to absorption changes, which occur in response to dietary manganese level (Suso and Edwards, 1969). The observed reduction in liver accumulation is accompanied by an increased rate of accumulation in muscle between 20G0 and 3000 ppm dietary Mn. This supports other studies that indicated that the capacity of liver
TABLE 6. Effect of dietary manganese (Mn) and age on the mineral composition of plasma in chicks Mairl treatment effects
Treaa • n e n t Added Mn
Age
(ppm) 1
(weeks)
Mineral concentratic in (Mg/dl) Mn Zinc Copper
Manganese level effects2 0
1000 2000 3000 SE3
4.18 6.07 7.96 11.95
140 184 182 182
16.0 21.3 24.7 25.7
.70
10
2.2
Age effects" 1 2 3 SE3
9.08 7.58 5.97 .60
194 174 148 9
24.3 19.4 22.1 1.9
Statistical significance Mn Age
<.001 <.05
<.05 <.01
<.05 NSS
1 Basal diet contained 112 ppm. Supplemental Mn as reagent grade MnSO„ - H 2 0 . 2 Each value represents the mean of six pens, pooled from 2 chicks per pen.
'Pooled standard error. "Each value represents the mean of eight pens, pooled from 2 chicks per pen. 5
Nonsignificant (P>.05).
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Tissue
Correlation coefficient
691
692
BLACK ET AL. TABLE 7. Coefficient of variation and lambda criterion values of chick tissues as related to age Tissue
Age
Liver
Kidney
Pancreas
Muscle
Bone
Plasma
76.3 73.2 71.8
36.5 52.2 46.2
(weeks) Coefficient of variation1 1 2 3
32.1 26.0 24.4
39.6 45.7 46.0
49.0 52.9 50.4
35.3 41.0 57.1
1 2 3
6.95 6.87 5.75
7.72 7.41 8.49
6.00 5.72 7.56
4.18 5.00 4.63
7.91 8.42 8.36
1
Coefficient of variation = (SD/mean) X 100.
2
Lambda criterion values = slope/SD. For convenience, values have been multiplied by 10 4 .
to excrete Mn can become overloaded, leading to accumulation in other tissues (Hall et al, 1981). Manganese concentrations in kidney, pancreas, muscle, and plasma were reduced with age, whereas that in bone was increased and that in liver was not affected. Such reductions in soft tissue have been associated with dilution by growth of the organs (Rehnberg et al, 1981), changes in blood flow to the organ and permeability of cellular membranes (Task Group on Metal Accumulation, 1973), reduced intestinal absorption of particulate matter (Le Feure and Joel, 1977), development of excretory mechanism (Miller et al, 1975), and reduced tissue retention (Mena, 1981). Level of dietary Mn influenced tissue concentration of several trace minerals. The mutual antagonism of Mn and Fe has been documented (Diez-Ewald et al, 1968). Liver and pancreas Fe were reduced by dietary Mn. Plasma and kidney Zn were increased by dietary Mn. Ivan and Grieve (1975) noted an increased liver, kidney and heart Zn in Mn-supplemented calves. In contrast, Hamilton et al. (1979) found supplemental Zn reduced Mn in the liver of Japanese quail. Dietary Mn increased Cu in the present study in liver and plasma. This agrees with other reports of increased liver (Anke et al, 1973) and plasma Cu (Gubler et al., 1954) in Mn-supplemented goats and rats, respectively. It has been demonstrated (Black et al, 1984a,b) that tissue accumulation of Mn at high dietary intakes is a sensitive bioassay for
7.55 7.28 7.89
Mn in chicks. Because there is a difference in absorption and tissue level with age, it was of interest to determine the age that provided the most sensitive bioassay. To evaluate age differen ces, coefficient of variation [CV = (SLV mean) X 100] and X criterion values (X = slope/SD) were determined (Table 7) for various tissues at 1, 2, and 3 weeks of age. In kidney and muscle, CV increased with age, while in bone and liver it decreased with no pattern observed in pancreas or plasma. The X values were higher for all tissues except liver at 3 weeks compared to 1 week. It had been determined previously (Black et al., 1984a) that bone and kidney provided the most sensitive bioassay for Mn. The highest X values were obtained with kidney at 3 weeks followed by bone at 2 weeks. These data suggest, based on X criterion values, that a 3-week bioassay study may provide better differentiation among sources than a shorter trial. REFERENCES Anke, M., A. Hennig, B. Groppel, G. Dittrich, and M. Grun, 1973. Manganmangel beim Wiederkauer. 4. Der Einfluss des Manganmangels auf den Gehalt neugeborener Lammer an Fett, Protein, Mangan, Asche, Kalzium, Phosphor, Zink and Kupfer. Arch. Tierernaehr. 23:213-223. Anonymous, 1974. Analytical Methods for Atomic Absorption Spectroscopy Using the HGA Graphite Furnace. The Perkin-Elmer Corp., Norwalk, CT. Anonymous, 1982. Analytical Methods for Atomic Absorption Spectrophotometry. The PerkinElmer Corp., Norwalk, CT. Black, J. R., C. B. Ammerman, P. R. Henry, and R. D. Miles, 1984a. Tissue manganese uptake as a
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Lambda criterion values2
MANGANESE BIOAVAILABILITY
1403-1408. Maynard, L. S., and G. C. Cotzias, 1955. The partition of manganese among organs and intracellular organelles of the rat. J. Biol. Chem. 2 1 4 : 4 8 9 495. Mena, I., 1981, Manganese. Pages 2 3 3 - 2 7 0 in Disorders of Mineral Metabolism. Vol. I. F. Bronner and J. W. Coburn, ed. Academic Press, New York, NY. Miller, S. T., G. C. Cotzias, and H. A. Evert, 1975. Control of tissue manganese: Initial absence and sudden emergence of excretion in the neonatal mouse. Am. J. Physiol. 229:1080-1084. Rehnberg, G. L., J. F. Hein, S. D. Carter, R. S. Linko, and J. W. Laskey, 1981. Chronic ingestion of M n 3 0 4 by young rats: tissue accumulation, distribution, and depletion. J. Toxicol. Environ. Health 7:263-272. Southern, L. L., and D. H. Baker, 1983. Excess manganese ingestion in the chick. Poultry Sci. 62:642-646. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics: A Biometrical Approach. 2nd ed. McGraw-Hill Book Co., New York, NY. Suso, F. A., and H. M. Edwards, Jr, 1969. Whole body counter studies on the absoprtion of 6 0 Co, 59 Fe, 54 Mn, and 6 s Zn by chicks, as affected by their dietary levels and other supplemental divalent elements. Poultry Sci. 48:933-938. Task Group on Metal Accumulation, 1973. Accumulation of toxic metals with special reference to their absorption, excretion and biological halftimes. Environ. Physiol. Biochem. 3:65—107. Underwood, E. J., 1977. Trace Elements in Human and Animal Nutrition. 4th ed. Academic Press, New York, NY.
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measure-of manganese bioavailability. Nutr. Rep. Int. 29:807-814. Black, J. R., C. B. Ammerman, P. R. Henry, and R. D. Miles, 1984b. Biological availability of manganese sources and effects of high dietary manganese on tissue mineral composition of broiler-type chicks. Poultry Sci. 63:1999-2006. Cahill, D. F., M. S. Bercegeay, R. C. Haggerty, J. E. Gerding, and L. E. Gray, 1980. Age-related retention and distribution of ingested M n 3 0 4 in the rat. Toxico. Appl. Pharmacol. 5 3 : 8 3 - 9 1 . Diez-Ewald, M., L. R. Weintraub, and W. H. Crosby, 1968. Interrelationshp of iron and manganese metabolism. Proc. Soc. Exp. Biol. Med. 129: 448-455. Gubler, C. J., D. S. Taylor, E. J. Eichwald, G. E. Cartwright, and M. M. Wintrobe, 1954. Copper metabolism. XII. Influence of manganese on metabolism of copper. Proc. Soc. Exp. Biol. Med. 86:223-227. Hall, E. D., H. W. Symonds, and W. M. Allen, 1981. The roles of the liver and gut in Mn homeostasis in cattle. Pages 89—91 in Trace Element Metabolism in Man and Animals — IV. J. McC. Howell, J. M. Gawthorne and C. L. White, ed. Aust. Acad. Sci., Canberra. Hamilton, R. P., M. R. S. Fox, B. E. Fry, Jr., A.O.L. Jones, and R. M. Jacobs, 1979. Zinc interference with copper, iron and manganese in young Japanese quail. J. Food Sci. 44:738—741. Ivan, M., and C. M. Grieve, 1975. Effects of zinc, copper, and manganese supplementation of high-concentrate ration on digestibility, growth, and tissue content of Holstein calves. J. Dairy Sci. 58:410-415. Le Feure, M. E., and D. D. Joel, 1977. Intestinal absorption of particulate matter. Life Sci. 21:
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