Bioavailability and toxicity of manganese in ruminants and poultry

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Bioavailability and toxicity of manganese in ruminants and poultry
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Black, James Robert, 1950-
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Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
        Page iv
    Table of Contents
        Page v
        Page vi
        Page vii
    List of Tables
        Page viii
        Page ix
        Page x
    List of Figures
        Page xi
        Page xii
        Page xiii
    Abstract
        Page xiv
        Page xv
    Chapter 1. Introduction
        Page 1
        Page 2
        Page 3
    Chapter 2. Review of literature
        Page 4
        Page 5
        Page 6
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    Chapter 3. Biological availability of reagent grade manganese sources and effects of high dietary manganese in broiler-type chicks--Experiment 1
        Page 47
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    Chapter 4. Effects of high, graded dietary levels of reagent grade manganese monoxide on tissue mineral composition of broiler-type chicks--Experiment 2
        Page 75
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    Chapter 5. Effects of dietary manganese level and age on tissue mineral composition of broiler-type chicks supplemented with reagent grade manganese sulfate monohydrate--Experiment 3
        Page 94
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    Chapter 6. Effects of high dietary manganese from feed grade manganese oxide and reagent grade manganese carbonate in sheep--Experiment 4
        Page 119
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    Chapter 7. Effects of level and route of administration of manganese from reagent grade manganese monoxide on intake and blood parameters in sheep--Experiment 5
        Page 147
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    Chapter 8. Influence of high dietary manganese from reagent grade manganese monoxide on tissue manganese accumulation and depletion and tissue mineral composition--Experiment 6
        Page 156
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    Chapter 9. General conclusions
        Page 177
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    Appendix. Supplemental tables
        Page 180
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    Bibliography
        Page 183
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    Biographical sketch
        Page 205
        Page 206
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        Page 208
Full Text












BIOAVAILABILITY AND TOXICITY OF MANGANESE
IN RUMINANTS AND POULTRY




By

JAMES ROBERT BLACK


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA


1983


























this

work is

dedicated

to these three . .

Honey

Elizabeth

and

Cheri














ACKNOWLEDGEMENTS


Sincere appreciation is extended to Dr. Clarence B. Ammerman,

chairman of the supervisory committee, for guidance, support, construc-

tive criticism, and for sharing his knowledge of mineral nutrition

throughout the academic program, experimental investigation, and pre-

paration of this dissertation. The author gratefully acknowledges the

assistance and time provided by other members of the supervisory commit-

tee including Drs. Richard D. Miles, Lee R. McDowell, Barney Harris,

Jr., and William G. Boggess. Appreciation is extended to Drs. Frank G.

Martin, IFAS Statistics, and Spencer M. Free, SmithKline Company,

Philadelphia, Pennsylvania, for assistance in statistical analysis, and

to Dr. James E. Marion for his supportive advice.

The author is grateful for the technical assistance of Pamela Henry

Miles and the assistance of John Funk, Nancy Wilkinson, Merle Smith, and

other members of the Nutrition Laboratory staff. Sincere appreciation

is expressed to Jack Stokes, Gary Russell, and others who assisted in

necropsy.

Acknowledgement is made to International Minerals and Chemical

Corporation, Mundelein, Illinois; Moorman Manufacturing Company, Quincy,

Illinois; Occidental Chemical Company, Houston, Texas; and Southeastern

Minerals, Inc., Bainbridge, Georgia, for financial support of this re-

search. The provision of experimental supplies by Monsanto Company,

St. Louis, Missouri; and Pfizer, Inc., New York, New York, is gratefully

acknowledged.









Appreciation is extended to V.M.S., Inc., Montgomery, Alabama;

the National Feed Ingredients Association, Des Moines, Iowa; the Florida

Citrus Processor's Association, Winter Haven, Florida; and the Grady E.

Black Professional Corporation, Griffin, Georgia, for personal financial

support.

Sincere appreciation is expressed to the author's wife, Cheri, for

assistance in laboratory analysis and to Adele Koehler for typing this

dissertation.

The author expresses special thanks to his parents for their love,

encouragement, advice, support, and personal sacrifice and to his wife

for her understanding, forbearance, inspiration, and love.














TABLE OF CONTENTS


ACKNOWLEDGEMENTS ....... ........................

LIST OF TABLES ........ .........................

LIST OF FIGURES ....... ........................

ABSTRACT ......... ...........................

CHAPTER

I INTRODUCTION ....... ......................

II REVIEW OF LITERATURE ...... .................

Manganese Functions and Manifestations of Deficiency .

Manganese and Bone Development ..............
Neonatal Ataxia and Development of the Inner Ear.
Manganese and Enzyme Activity ... ...........
Manganese and Reproduction ................
Carbohydrate and Lipid Metabolism ...........

Manganese Metabolism ..... ..................

Homeostasis ...... ....................
Absorption and Excretion .... .............
Blood Clearance, Tissue Retention, and Turnover .

Manganese and Mineral Interactions ... ...........

Iron ....... ........................
Calcium and Phosphorus .... ..............
Copper and Zinc ...... .................
Magnesium and Other Elements ...............

Manganese Requirements ..... .................
Manganese Bioavailability .... ................
Manganese Toxicosis ...... ..................


Pae


.viii

* xi

* xiv











III BIOLOGICAL AVAILABILITY OF REAGENT GRADE MANGANESE SOURCES
AND EFFECTS OF HIGH DIETARY MANGANESE IN BROILER-TYPE
CHICKS--EXPERIMENT 1 ...... .. .................... 47

Introduction. ........................47
Literature Review ...... ..................... 48
Materials and Methods ..... ................... ....50
Results ...... ... .......................... ...52
Discussion ...... .... ......................... 54

IV EFFECTS OF HIGH, GRADED DIETARY LEVELS OF REAGENT GRADE
MANGANESE MONOXIDE ON TISSUE MINERAL COMPOSITION OF
BROILER-TYPE CHICKS--EXPERIMENT 2 ... ............. ....75

Introduction. ......................................... 75
Literature Review ..... .. ................... 76
Materials and Methods ..... ................... ....77
Results ...... ... .......................... ...78
Discussion ...... .. ......................... ... 80

V EFFECTS OF DIETARY MANGANESE LEVEL AND AGE ON TISSUE
MINERAL COMPOSITION OF BROILER-TYPE CHICKS SUPPLEMENTED
WITH REAGENT GRADE MANGANESE SULFATE MONOHYDRATE--
EXPERIMENT 3 ...... .... ........................ 94

Introduction. ..................................... .. 94
Literature Review ..... ... .................. 95
Materials and Methods ..... ................... ....96
Results ..... .... .......................... ...98
Discussion ...... .. ......................... ...100

VI EFFECTS OF HIGH DIETARY MANGANESE FROM FEED GRADE
MANGANESE OXIDE AND REAGENT GRADE MANGANESE CARBONATE
IN SHEEP--EXPERIMENT 4 ..... ................... ...119

Introduction. .................................... 119
Literature Review ...... .................... ...120
Materials and Methods ...... ................... ...121
Results and Discussion ..... ................... ...122

VII EFFECTS OF LEVEL AND ROUTE OF ADMINISTRATION OF MANGANESE
FROM REAGENT GRADE MANGANESE MONOXIDE ON INTAKE AND BLOOD
PARAMETERS IN SHEEP--EXPERIMENT 5 .... ............. ...147

Introduction ....... ........................ ...147
Literature Review ..... .. ..................... ...148
Materials and Methods ...... ................... ...148
Results and Discussion ..... ................... ...149











VIII INFLUENCE OF HIGH DIETARY MANGANESE FROM REAGENT GRADE
MANGANESE MONOXIDE ON TISSUE MANGANESE ACCUMULATION AND
DEPLETION AND TISSUE MINERAL COMPOSITION--EXPERIMENT 6.

Introduction ....... ......................
Literature Review ...... ....................
Materials and Methods ..... ..................
Results and Discussion ..... ..................

IX GENERAL CONCLUSIONS ...... ..................

APPENDIX


SUPPLEMENTAL TABLES .

BIBLIOGRAPHY ............

BIOGRAPHICAL SKETCH ........


. . . . . . .

. . . . . . .

. . . . . . .














LIST OF TABLES


Table Page

1. COMPOSITION OF BASAL DIET--EXP. 1, 2, AND 3 ............ 60

2. CHEMICAL AND PHYSICAL CHARACTERISTICS OF MANGANESE
SOURCES--EXP. 1-6 ......... ...................... 61

3. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON FEED
INTAKE, GAIN, AND FEED CONVERSION IN CHICKS--EXP. 1 ....... 62

4. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON HEMO-
GLOBIN, HEMATOCRIT, AND MINERAL COMPOSITION OF PLASMA IN
CHICKS--EXP. 1 ........ ....................... ..63

5. LINEAR REGRESSION ANALYSIS OF TISSUE MANGANESE WITH
RESPECT TO DIETARY MANGANESE--EXP. 1 ..... ............ 64

6. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE
MINERAL COMPOSITION OF LIVER IN CHICKS--EXP. 1 ......... ...65

7. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE
MINERAL COMPOSITION OF HEART IN CHICKS--EXP. 1 ......... ...66

8. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE
MINERAL COMPOSITION OF MUSCLE IN CHICKS--EXP. 1 .......... 67

9. EFFECT OF DIETARY MANGANESE ON BONE ASH AND MINERAL
COMPOSITION OF BONE IN CHICKS--EXP. 1 ............... ...68

10. RELATIVE BIOLOGICAL AVAILABILITY OF THREE MANGANESE SOURCES
BASED ON MULTIPLE LINEAR REGRESSION, LINEAR REGRESSION, AND
TISSUE MANGANESE CONCENTRATION--EXP. 1 .............. ...69

11. EFFECTS OF DIETARY MANGANESE ON FEED INTAKE, GAIN, AND
FEED CONVERSION IN CHICKS--EXP. 2 .... .............. ..84

12. EFFECT OF DIETARY MANGANESE ON THE MINERAL COMPOSITION OF
LIVER, KIDNEY, AND MUSCLE IN CHICKS--EXP. 2 ........... ...85

13. LINEAR REGRESSION ANALYSIS OF TISSUE MANGANESE UPTAKE WITH
RESPECT TO DIETARY MANGANESE FROM HIGH, GRADED LEVELS OF
REAGENT GRADE MANGANOUS MONOXIDE--EXP. 2 ............. ..86

14. EFFECT OF DIETARY MANGANESE ON BONE ASH AND MINERAL COM-
POSITION OF BONE AND PLASMA IN CHICKS--EXP. 2 ........... 87









Table Page

15. LAMBDA CRITERION VALUES AND LINEAR REGRESSION CORRELATION
COEFFICIENTS FOR TISSUE UPTAKE OF MANGANESE IN CHICKS--
EXP. 2 ......... ............................ 88

16. EFFECTS OF LEVEL OF DIETARY MANGANESE AND AGE ON FEED
INTAKE, GAIN, AND FEED CONVERSION IN CHICKS--EXP. 3 ....... 104

17. EFFECTS OF LEVEL OF DIETARY MANGANESE AND AGE ON THE
MINERAL COMPOSITION OF LIVER IN CHICKS--EXP. 3 ........... 105

18. MULTIPLE LINEAR REGRESSION ANALYSIS OF TISSUE MANGANESE
WITH RESPECT TO DIETARY MANGANESE AND AGE--EXP. 3 ........ 106

19. EFFECTS OF LEVEL OF DIETARY MANGANESE AND AGE ON THE
MINERAL COMPOSITION OF KIDNEY IN CHICKS--EXP. 3 ......... ..107

20. EFFECTS OF LEVEL OF DIETARY MANGANESE AND AGE ON THE
MINERAL COMPOSITION OF PANCREAS IN CHICKS--EXP. 3 ........ 108

21. EFFECTS OF LEVEL OF DIETARY MANGANESE AND AGE ON THE
MINERAL COMPOSITION OF MUSCLE IN CHICKS--EXP. 3 ......... ..109

22. EFFECTS OF LEVEL OF DIETARY MANGANESE AND AGE ON BONE ASH AND
BONE MINERAL COMPOSITION IN CHICKS--EXP. 3 ............. .110

23. EFFECTS OF LEVEL OF DIETARY MANGANESE AND AGE ON THE
MINERAL COMPOSITION OF PLASMA IN CHICKS--EXP. 3 ......... ..111

24. COEFFICIENT OF VARIATION AND x CRITERION VALUES OF CHICK
TISSUES AS RELATED TO AGE--EXP. 3 .... .............. .112

25. COMPOSITION OF BASAL DIET--EXP. 4, 5, 6 ............. ..129

26. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON FEED
INTAKE, GAIN, AND FEED CONVERSION IN SHEEP--EXP. 4 ........ 131

27. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON
HEMOGLOBIN, HEMATOCRIT, AND MINERAL COMPOSITION OF SERUM
IN SHEEP--EXP. 4 ....... ....................... ..132

28. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE
MINERAL COMPOSITION OF LIVER IN SHEEP--EXP. 4 ... ........ 133

29. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE
MINERAL COMPOSITION OF KIDNEY IN SHEEP--EXP. 4 ........... 134

30. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE
MINERAL COMPOSITION OF SPLEEN IN SHEEP--EXP. 4 ........... 135

31. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE
MINERAL COMPOSITION OF HEART IN SHEEP--EXP. 4 .......... .136









Table Page

32. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE
MINERAL COMPOSITION OF SKELETAL MUSCLE IN SHEEP--EXP. 4 . 137

33. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE
MINERAL COMPOSITION OF BONE IN SHEEP--EXP. 4 ........... ..138

34. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON PER-
CENTAGE BONE ASH IN SHEEP--EXP. 4 .... .............. .139

35. MULTIPLE LINEAR REGRESSION ANALYSIS OF TISSUE MANGANESE
WITH RESPECT TO DIETARY MANGANESE FROM TWO SOURCES--EXP. 4.. 140
36. EFFECTS OF LEVEL AND ROUTE OF ADMINISTRATION OF MANGANESE
ON FEED INTAKE OF SHEEP FOR THREE ONE-WEEK INTERVALS--
EXP. 5 ...... ... ............................ .152

37. EFFECTS OF LEVEL AND ROUTE OF ADMINISTRATION OF MANGANESE
ON HEMOGLOBIN, HEMATOCRIT, AND SERUM MANGANESE IN SHEEP--
EXP. 5 ......... ............................ .153

38. EFFECT OF MANGANESE ON HEMOGLOBIN, HEMATOCRIT, AND MINERAL
COMPOSITION OF SERUM FROM SHEEP DURING CONTROL, ACCUMULA-
TION, AND DEPLETION PHASES--EXP. 6 .... .............. ..165

39. EFFECT OF DIETARY MANGANESE ON TISSUE MINERAL COMPOSITION
OF SHEEP DURING CONTROL, ACCUMULATION, AND DEPLETION
PHASES--EXP. 6 ........ ........................ .166

40. EFFECT OF DIETARY MANGANESE ON MUSCLE MINERAL COMPOSITION
OF SHEEP DURING CONTROL, ACCUMULATION, AND DEPLETION
PHASES--EXP. 6 ........ ........................ .167

41. EFFECT OF DIETARY MANGANESE ON BONE ASH AND MINERAL COM-
POSITION OF SHEEP DURING CONTROL, ACCUMULATION, AND
DEPLETION PHASES--EXP. 6 ...... ................... ..168

A-1. A COMPARISON OF PREPARATION REQUIRED FOR SOFT TISSUE AND
BONE PRIOR TO MINERAL ANALYSIS ..... ................ .181

A-2. EFFECTS OF LEVEL OF DIETARY MANGANESE AND AGE ON THE
COPPER CONCENTRATION OF KIDNEY IN CHICKS--EXP. 3 .......... 182














LIST OF FIGURES


Figure Page
1. Influence of homeostasis on tissue mineral concentration
with respect to dietary intake level .... ............ 21
2. Effect of level and source of dietary Mn on plasma Mn
concentration in chicks--Exp. 1 .................. ...70
3. Effect of level and source of dietary Mn on liver Mn
concentration in chicks--Exp. 1 ..... ............... 71
4. Effect of level and source of dietary Mn on heart Mn
concentration in chicks--Exp. 1 .................. ...72
5. Effect of level and source of dietary Mn on muscle Mn
concentration in chicks--Exp. 1 .................. ...73
6. Effect of level and source of dietary Mn on bone Mn
concentration in chicks--Exp. 1. .................... 74
7. Measured and predicted Mn concentrations of liver with
respect to dietary intake of reagent grade MnO in chicks--
Exp. 2 ...... ... ........................... 89

8. Measured and predicted Mn concentrations of kidney with
respect to dietary intake of reagent grade MnO in chicks--
Exp. 2 ...... ... ........................... 90

9. Measured and predicted Mn concentrations of muscle with
respect to dietary intake of reagent grade MnO in chicks--
Exp. 2 ...... ... ........................... 91

10. Measured and predicted Mn concentrations of bone with
respect to dietary intake of reagent grade MnO in chicks--
Exp. 2 ...... ... ........................... 92

11. Measured and predicted Mn concentrations of plasma with
respect to dietary intake of reagent grade MnO in chicks--
Exp. 2 ...... ... ........................... 93

12. Effect of level of dietary Mn from reagent grade MnO and
age on liver Mn concentration in chicks--Exp. 3 .......... 113

13. Effect of level of dietary Mn from reagent grade MnO and
age on kidney Mn concentration in chicks--Exp. 3 ........ 114









Figure Page

14. Effect of level of dietary Mn from reagent grade MnO and
age on pancreas Mn concentration in chicks--Exp. 3 ......... 115

15. Effect of level of dietary Mn from reagent grade MnO and
age on muscle Mn concentration in chicks--Exp. 3 .......... 116
16. Effect of level of dietary Mn from reagent grade MnO and
age on bone Mn concentration in chicks--Exp. 3 ........... 117
17. Effect of level of dietary Mn from reagent grade MnO and
age on plasma Mn concentration in chicks--Exp. 3 .......... 118

18. Effect of level and source of dietary Mn on serum Mn
concentration in sheep--Exp. 4 ..... ................ .141
19. Effect of level and source of dietary Mn on liver Mn
concentration in sheep--Exp. 4 ...... ................ 142
20. Effect of level and source of dietary Mn on kidney Mn
concentration in sheep--Exp. 4 ...... ................ 143
21. Effect of level and source of dietary Mn on spleen Mn
concentration in sheep--Exp. 4 ..... ................ .144
22. Effect of level and source of dietary Mn on cardiac and
skeletal muscle Mn concentration in sheep--Exp. 4 ........ 145
23. Effect of level and source of dietary Mn on bone Mn
concentration in sheep--Exp. 4 ...... ................ 146

24. Effect of level of reagent grade MnO administered in the
diet on feed intake of sheep--Exp. 5 ................ .154
25. Effect of level of reagent grade MnO administered in
capsules on feed intake in sheep--Exp. 5 .... ........... 155

26. Effect of dietary Mn as reagent grade MnO on hemoglobin
and hematocrit in sheep--Exp. 6 ...... ............... 169

27. Effect of dietary Mn as reagent grade MnO on serum Mn
accumulation and depletion in sheep--Exp. 6 ........... .170

28. Effect of dietary Mn as reagent grade MnO on liver Mn
accumulation and depletion in sheep--Exp. 6 ........... .171

29. Effect of dietary Mn as reagent grade MnO on kidney Mn
accumulation and depletion in sheep--Exp. 6 ........... ..172

30. Effect of dietary Mn as reagent grade MnO on spleen Mn
accumulation and depletion in sheep--Exp. 6 ........... .173








Figure Page

31. Effect of dietary Mn as reagent grade MnO on heart Mn
accumulation and depletion in sheep--Exp. 6 ... ......... 174

32. Effect of dietary Mn as reagent grade MnO on skeletal
muscle Mn accumulation and depletion in sheep--Exp. 6 . . 175

33. Effect of dietary Mn as reagent grade MnO on bone Mn
accumulation and depletion in sheep--Exp. 6 ... ......... 176









Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy

BIOAVAILABILITY AND TOXICITY OF MANGANESE
IN RUMINANTS AND POULTRY

by

James Robert Black

December 1983

Chairman: Clarence B. Ammerman
Major Department: Animal Science

Six experiments were conducted to investigate effects of high levels

of dietary Mn on performance, tissue Mn concentration, and tissue mineral

composition in chicks and sheep. The Mn sources included reagent grade

(rg) MnO, MnSO4.H20, and MnCO3 and feed grade (fg) MnO. In addition, a

new inorganic Mn bioassay was proposed. In Experiment 1 chicks were fed

diets supplemented at levels to 4000 ppm from rg MnSO4.H20, MnO, and

MnCO3 to observe effects on performance and compare tissue accumulation

from different sources as a bioassay. There was no effect on feed in-

take, weight gain, or feed conversion. The bioassay indicated that

MnSO4.H20 was most available, followed by MnO and MnCO3 was least avail-

able. The relationship between dietary Mn and tissue Mn and performance

in chicks fed graded levels of rg MnO to 3000 ppm was investigated in

Experiment 2. Tissue Mn increased linearly as dietary level increased

and performance was not affected. The bioassay was evaluated in Experi-

ment 3 with chicks fed up to 3000 ppm as rg MnSO4.H20 for 1, 2, or 3

weeks to determine if age influenced sensitivity of the bioassay and

observe signs of toxicosis. The three-week bioassay was slightly more

sensitive but data indicated that a one-week bioassay might be satis-

factory. Experiment 4 was conducted with sheep to investigate long-term









dietary effects of Mn as fg MnO up to 4000 ppm and rg MnCO3 up to 8000

ppm. Feed intake was reduced by high levels from both sources. Experi-

ment 5 was conducted to demonstrate whether the toxic effect of de-

creased feed intake was a physiological or palatability response by

feeding rg MnO in the diet or capsule. Data indicated the response was

physiological in nature. In Experiment 6 sheep were fed a diet supple-

mented with 8000 ppm Mn for 42 days, then a normal diet for 84 days to

measure tissue accumulation and depletion of Mn from rg MnO. Tissue Mn

increased during accumulation and reached normal levels within three

weeks of Mn withdrawal. Several tissue minerals were affected by the

high level of dietary Mn.














CHAPTER I

INTRODUCTION


Manganese (Mn) is an essential trace element that is distributed

widely throughout nature. It is found in igneous, sedimentary, and

metamorphic rocks, and is the principal metallic constituent of nodules

and other surface deposits in large areas of the deep ocean floors.

Approximately 1000 ppm of the earth's crust is comprised of Mn with

soil content ranges reported to be from less than 1 to as much as

7000 ppm with an average of approximately 500 to 600 ppm (National
Academy of Sciences, 1973). Animal feeds of plant origin vary con-

siderably in Mn content from as little as 3 ppm in corn silage to over

800 ppm in some grasses and over 3000 ppm has been reported in white

lupin seeds. Protein concentrates of animal origin are much poorer

sources of Mn (5 to 15 ppm) while milk and milk products are quite low

(Underwood, 1981).
Inorganic Mn sources include ores, feed grade and reagent grade

Mn salts. Natural Mn ores include Mn in several forms: oxides--

manganosite [MnO], pyrolusite [MnO2], braunite [Mn203], hausmannite

[Mn304]; hydroxides--pyrochroite [Mn(OH)2], manganite [MnO(OH)];

carbonate--rhodochrosite [MnC03]; sulfate--szmikite [MnSO4-H20]; sul-

fides--halabandite [MnS], hauerite [MnS2]; silicates--tephroite [Mn2SiO],
rhodonite [MnSiO3]; chloride--scacchite [MnCl2]; and pyrophosphate--
reddingite [Mn3(PO4)2.3H20] (CRC, 1979). Feed grade sources are








manganous oxide (MnO), manganese sulfate (MnSO4.H20), and manganese

carbonate (MnCO3). Under some conditions, feed grade MnO can oxidize

and will therefore contain mixtures of MnO and the less available higher

oxidation forms. Manganous oxide is the preferred and most commonly fed

source, although in deciding which Mn compound to use, one must take

into consideration relative biological availability, freedom from con-

taminants such as heavy metals, and the relative economics of the com-

pounds (DeCreane, 1983).

Since Bertrand and Medigreceanu (1912) first reported traces of

Mn in tissues of approximately 60 wild and domestic animals and postu-

lated that its presence had some physiological significance, Mn in

animal tissues has been found to be essential for normal growth, skeletal

and otolith development, reproduction, carbohydrate and lipid metabolism,

and development and function of the central nervous system. Over 50

years ago, two groups of researchers working independently demonstrated

the essential nature of Mn for growth and fertility in mice (Kemmerer

et al., 1931) and rats (Orent and McCollum, 1931). Subsequently, Mn

was shown to have practical significance in that two diseases of poultry

known as perosis or "slipped tendon" (Wilgus et al., 1936, 1937a) and

nutritional chondrodystrophy (Lyons and Insko, 1937) were found to be

due to a dietary deficiency of this element and to be preventable by

Mn supplementation. Manganese deficiency was not demonstrated in

ruminants until 1951 by Bentley and Phillips.

High levels of Mn administered through different routes have pro-

duced Mn toxicosis in several species. It is well tolerated at dietary

levels of more than 1000 ppm in most species (National Research Council,

1980). The Select Committee on GRAS Substances (1979) of the Life





-3-


Sciences Research Office reported in an extensive review that while

MnO comprises 90% of the Mn supplemented in animal feeds, there are

insufficient data upon which to evaluate it as a Generally Recognized

as Safe (GRAS) feed ingredient. They cite the need for acute oral

toxicity studies and long-term feeding studies of MnO.

The experiments reported herein were conducted to evaluate effects

of high dietary levels of feed grade and reagent grade MnO, and reagent

grade MnCO3 and MnSO4.H20. In addition, a new method for the deter-

mination of relative biological availability of inorganic Mn sources

has been proposed.














CHAPTER II

LITERATURE REVIEW


Manganese is a group VII transition element that is found through-

out the biosphere. It has been calculated to be the twelfth most abun-

dant element in the earth's crust. The most stable salts are divalent

although it can have valences of -3, -1, 0, 1, 2, 3, 4, 5, 6, and 7.

Manganese was first recognized as an element by Scheele, Bergman, and

others and was first isolated by Gahn in 1774 by reduction of the dioxide

by carbon (National Academy of Sciences, 1973). Manganese occurs in

minute concentrations within the cells of all living things and has

been established as essential to a wide variety of organisms ranging

from bacteria to plants and animals (Williams, 1967).

The average Mn concentration in animal tissues is less than 1 ppm

and this small quantity is widely, though unevenly, distributed throughout

tissues and fluids. Tissue Mn concentration has been observed to be

characteristic of individual organs and independent of species (Cotzias,

1958). Bertrand and Medigreceanu (1912) reported finding trace amounts

of Mn in approximately 60 wild and domestic animal species and postu-

lated that its presence had some physiological significance and was not

merely a contaminant, as was commonly supposed. Twenty years later two

independent groups of researchers demonstrated the essential nature of

Mn; for growth and ovarian function in mice (Kemmerer et al., 1931) and

for prevention of testicular degeneration in rats (Orent and McCollum,

1931).









Manganese Functions and Manifestations of Deficiency


Manganese deficiency has been demonstrated in several species

including mice, rats, rabbits, guinea pigs, swine, poultry, cattle,

sheep, and goats (Underwood, 1977) and has been associated with a vita-

min K deficiency in man (Doisy, 1974). It was shown to have practical

significance when two diseases of poultry, perosis or "slipped tendon"

(Wilgus et al., 1936, 1937a) and nutritional chondrodystrophy (Lyons and

Insko, 1937), were found to be due to a deficiency of this mineral and

to be preventable by Mn supplementation. Perosis is characterized by

lameness resulting from enlargement and malformation of the tibiometa-

tarsal joint, thickening and shortening of the long bones, twisting and

bending of the tibia, and slipping of the gastrocnemius tendon from its

chondyles. Nutritional chondrodystrophy is due to deficiency in the

chick embryo and is characterized by short, thick legs and wings, shorten-

ing of the lower mandible ("parrot beak"), a skull that protrudes

anteriorly, and results in a high mortality rate.

The name perosis was first proposed by Titus and Ginn (1931) and

Titus (1932) who found that rice bran contained a factor that prevented

the disease. Heller and Penquite (1936, 1937) recognized a protective

factor in a water soluble extract of rice bran, while Sherwood and Fraps

(1936) reported that the ash of grey wheat shorts protected against

perosis and concluded that the factor was inorganic in nature. Wilgus

et al. (1936, 1937a) demonstrated that perosis-preventing properties of

cereals are directly related to their Mn content.

The primary manifestations of Mn deficiency were found in all

species studied and included impaired growth, skeletal abnormalities,









disturbed or depressed reproductive function, postural defects, ataxia

in the young, and defects in carbohydrate and lipid metabolism. These

deficiency signs vary with species, age, stage of growth, and degree and

severity of deficiency (Underwood, 1977). Although the identification

of a specific biochemical role for Mn proved elusive for many years,

many of the gross effects of Mn deficiency can be explained in terms of

its effect on mucopolysaccharide synthesis as explained in the following

section.


Manganese and Bone Development


The skeletal abnormalities associated with Mn deficiency in chicks

have also been reported in turkeys (Evans et al., 1942), swans (Emmel,

1944), and ducklings (Bernard and Demers, 1953). Shortening and bowing

of the forelegs was particularly characteristic of Mn deficiency in rats

(Barnes et al., 1941), mice (Shils and McCollum, 1943), guinea pigs

(Everson et al., 1959), and rabbits (Smith et al., 1944; Ellis et al.,

1947). Deficiency in cows produced calves which exhibited enlarged

knee and pastern joints with twisted legs (Rao, 1963) and difficulty

standing (Howes and Dyer, 1971). It has been implicated in "overknuck-

ling," a disease characterized by bone deformity and poor growth in

cattle raised on certain pastures in Holland (Grashuis et al., 1953)

although Hartmans (1974) suggested that this may be a conditioned response

brought on by high concentration of Fe, Ca, P, and K in the diets of these

animals. A deficiency in pigs was expressed by enlarged hock joints,

with crooked, shortened legs and lameness (Miller et al., 1940). Hurley

and Asling (1963) described a marked epiphyseal dysplasia of the proximal









tibial epiphysis and Hurley et al. (1960) reported an anomalous develop-

ment of ossification in the inner ear of Mn-deficient rats. The same

group (Hurley et al., 1961) reported disproportionate growth in the

long bones of Mn-deficient rats.

The observations concerning bone abnormalities and initial reports

of low blood and bone alkaline phosphatase concentration in deficient

animals (Weise et al., 1939) directed a search for a role of Mn in the

calcification process. Much of the evidence did not support such a

hypothesis. Investigations by Gallup and Norris (1938) and Caskey et

al. (1939) revealed thickened, shortened bones with slight reductions

in ash content in Mn-deficient chicks. Based on X-ray examination and

AgNO3 staining, however, calcification appeared to be normal. Parker

and coworkers (1955) indicated that dietary Mn level did not affect

amount or location of 45Ca and 32P deposited in the tibiae.

Histological changes observed in the epiphyseal cartilage of bones

from chicks with perosis turned attention to a possible involvement of

Mn in the synthesis of the organic matrix of cartilage (Wolbach and

Hegsted, 1953). Evidence that the primary changes in chondrogenesis

were in the epiphyseal plate led to the determination of the chemical

composition of this plate by Leach in 1960 who found a substantial in-

crease in fat and a decrease in hexuronic acid content in Mn deficiency.

Leach and Muenster (1962) reported a severe reduction in cartilage

mucopolysaccharide content and radiosulfate uptake in Mn-deficient

chicks. Total concentration of hexosamines and hexuronic acid were

reduced in cartilage and there was a less pronounced reduction of the

hexosamine content of other tissues. Chondroitin sulfate was the muco-

polysaccharide affected most severely by Mn deficiency. These findings









plus subsequent histological studies (Leach, 1968) supported the

hypothesis that Mn affects skeletal formation through chondrogenesis

rather than osteogenesis.

In studies with other species, similar effects upon the mucopoly-

saccharide content of cartilage were reported in guinea pigs (Tsai and

Everson, 1967; Shrader and Everson, 1967) and calves (Rojas et al.,

1965). Hyaluronic acid and heparin were also reduced. In addition to

the effect on skeletal development, Longstaff and Hill (1972) have re-

ported similar compositional changes in egg shell matrix which may explain

the earlier effects on egg shell formation described by Lyons (1939).

The impaired synthesis of mucopolysaccharides associated with Mn

deficiency has been linked to the activation of glycosyltransferases by

this element (Leach, 1971). This group of enzymes is important in poly-

saccharide and glycoprotein synthesis and Mn is usually the most effec-

tive of the metal ions required for their activity. Leach and coworkers

(1969) identified the critical sites of Mn function in chondroitin

sulfate synthesis that are defective in Mn deficient cartilage:

1. polymerase enzyme, which is responsible for the polymerization

of uridine diphosphate-N-acetylgalactosamine to uridine diphosphate-

N-acetylglucuronic acid, and is required for synthesis of the poly-

saccharide; and

2. galactotransferase, an enzyme that incorporates galactose from

uridine diphosphate-N-acetylgalactose into the galactose-galactose-

xylose trisaccharide which forms the linkage between the polysaccharide

and a serine of the associated protein.

These findings provide not only a biochemical explanation for the

defects observed in skeletal and connective tissue associated with Mn









deficiency, but also constitute the first established link between
effects of Mn deficiency observed in vivo and a specific underlying

biochemical defect.


Neonatal Ataxia and Development of the Inner Ear

Ataxia in the offspring of Mn-deficient animals, characterized by
incoordination, loss of equilibrium, head retraction, and high mortality,

was first observed in the chick by Caskey and Norris (1940) and in rats

by Shils and McCollum (1943). The association between Mn deficiency and

ataxia has subsequently been well established in rats (Hill et al.,

1950; Hurley et al., 1958), mice (Erway et al., 1970), guinea pigs
(Everson et al., 1959), pigs (Plumlee et al., 1956), and calves (Groppel

and Anke, 1971). Manganese supplementation after birth cannot reverse

ataxia and there is a critical time during gestation after which maternal

Mn supplementation is ineffective in preventing this lesion (Hurley et

al., 1958). There was evidence that the effect of Mn on mucopolysac-

charide metabolism was a causative factor in congenital ataxia observed

in the offspring of Mn-deficient animals. It was demonstrated in rats

(Asling et al., 1960), mice (Erway et al., 1970), and guinea pigs

(Shrader and Everson, 1967) that ataxia and postural defects arose from

impaired vestibular function, a reflection of the effect of Mn on

cartilage mucopolysaccharide synthesis and subsequent bone development

of the skull, particularly the otoliths of the utricular and saccular

maculae. These otoliths are characteristically absent or deformed in

Mn-deficient young.
A specific congenital ataxia in mice caused by the presence of a
mutant gene affecting coat color (pallid) results from defective









development of the otoliths due to impaired Mn metabolism (Hurley,

1968). Erway et al. (1966) have shown that the histologic changes and

ataxia can be completely prevented by the addition of 1000 ppm Mn to the

maternal diet. The expression of the mutant gene was prevented entirely.

This was the first demonstration that a phenocopy-inducing agent (in

this case Mn) can have a reciprocal effect on the gene itself. These

workers thought that the lesions may have been secondary to a lack of

normal pigmentation in the membranous labyrinth of pallid mice. Manganese

is much more abundant in pigmented tissue than in nonpigmented tissue

and Cotzias et al. (1964) have suggested that the pigmented tissue is

necessary to ensure an adequate local supply of Mn to the otoliths and

that high dietary levels were necessary to overcome the absence of such

pigmented tissue. Similar studies have reported that the occurrence of

a genetic postural defect, "screw neck," in pastel mink is associated

with defective otolith development and can be reduced with 1000 ppm Mn

(Erway and Mitchell, 1973).

Ataxia in the offspring of Mn-deficient mothers was initially

thought to be a result of brain dysfunction. This effect has clearly

been linked to defective otolith development. There is limited evidence

which indicates that Mn deficiency may result in impaired brain func-

tion. Hurley and coworkers (1963) reported that Mn-deficient rats were

more susceptible to convulsive states than normal rats. This effect was

independent of the presence of ataxia.


Manganese and Enzyme Activity

The relationship of Mn with enzymes is similar to other transition

elements in that it may be classified into two categories:





-11-


(a) metalloenzymes, and (b) metal-enzyme complexes. This type of
categorization is based upon affinity of the metal for the enzyme rather

than on a functional basis (Leach, 1976). Unlike other transition ele-

ments such as Fe, Cu, and Zn, the number of Mn metalloenzymes is limited,

whereas the enzymes that can be activated by Mn are numerous and include

hydrolases, kinases, decarboxylases, and transferases (Vallee and

Coleman, 1964). Many of these activations are nonspecific and Mn may

be partly replaced by other divalent metal ions, particularly Mg (O'Dell

and Campbell, 1971). This has contributed to the difficulty of deter-

mining specific biochemical roles for Mn. Two important enzymes that

demand Mn exclusively are the peptidase prolidase and succinic dehydrogen-

ase. Prolidase is an intestial digestive enzyme which hydrolyzes the

dipeptide glycylproline and succinic dehydrogenase is involved in the

citric acid cycle (Orten and Neuhaus, 1970).

The primary importance of polymerase and galactotransferase in

mucopolysaccharide synthesis as outlined by Leach and coworkers (1969)

has been discussed previously. Also mentioned was the earlier work of

Wiese et al. (1939) which reported decreased blood and bone phosphatase

activity in Mn-deficient chicks which initiated some research into a

possible role of Mn in osteogenesis. It was postulated that Mn might

serve as an activator for this enzyme. Subsequent studies on this effect

have been contradictory. Smith and Ellis (1947) reported reduced

activity of this enzyme in rabbits and these results were duplicated

in swine (Liebholz et al., 1962), sheep (Lassiter and Morton, 1968),

cattle (Rojas et al., 1965), and rats (Paynter, 1980). Two groups of

researchers found Mn deficiency in rats had no effect on phosphatase





-12-


activity (Wachtel et al., 1943; Hurley et al., 1959) while Everson and

associates (1959) found no effect in guinea pigs.

Pyruvate carboxylase isolated from chick mitochondria provided the

first clear evidence of a Mn metalloenzyme (Scrutton et al., 1966). This

enzyme was found in the mitochondria, contained 4 moles of tightly bound

Mn and 4 moles of biotin per mole of enzyme and catalyzed the conversion

of oxaloacetate to pyruvate. Studies by Mildvan et al. (1966) resulted

in the proposal that the electrophilic character of the bound Mn facili-

tated the proton departure from the methyl group of pyruvate and the

carboxyl transfer from the carboxybiotin residue to pyruvate. Even this

metalloenzyme was subject to partial substitution of Mg for Mn as de-

scribed by Scrutton et al. (1972) under conditions of Mn deficiency.

These workers found that the relative content of the two metals in the

enzyme was related to the severity of the deficiency with only minor

alterations in the catalytic properties.

In contrast to the observation with pyruvate carboxylase, Mn de-

ficiency resulted in a depletion of Mn from avimanganin, a protein of

unknown function isolated from avian liver, and a reduction in avi-

manganin content of liver (Scrutton, 1971). It appeared, therefore,

that the animal can adapt to Mn deficiency with respect to pyruvate

carboxylase by substituting Mg for Mn, whereas Mn deficiency limits the

production of the metalloprotein avimanganin. Manganese has been asso-

ciated with other enzymes involved in carbon dioxide fixation (Miller

et al., 1968).

A superoxide dismutase (SOD) isolated from chicken liver was

another metalloenzyme found to be activated by Mn (Weisiger and Fridovich,

1973). This enzyme is located in the mitochondrial matrix and catalyzes









the dismutation of the superoxide free radical (02") to H202 and H20
and is believed to protect the cell from the toxic effects of these

free radicals. Superoxide dismutase activity has been reduced by Mn

deficiency in rats (Paynter, 1980) and mice, rats, and chickens (de Rosa

et al., 1980). A different SOD found in the cytosol and intermembrane

space of mitochondria and activated by Cu or Zn increased in activity

concurrent to the reduction in Mn-SOD in the mitochondrial matrix of

Mn-deficient chickens. It was proposed that an intracellular accumula-

tion of 02 in response to decreased activity of Mn-SOD induced a com-

pensatory increase in the cytosolic form of this enzyme. Activity of

both forms returned to normal when a Mn-sufficient (1000 ppm) diet was

fed (de Rosa et al., 1980).

Deficiency of Mn was characterized by abnormalities in cell function

and ultrastructure, particularly to the mitochondrial membranes (Hurley

et al., 1970). Electron microscopy revealed alterations in cell mem-

brane integrity, swollen endoplasmic reticulum and irregular mitochon-

dria which contained elongated and stacked cristae (Bell and Hurley,

1973). De Rosa et al. (1980) postulated that the membrane damange ob-

served in Mn deficiency is due to the accumulation of free 02 generated

by respiratory activity inside the mitochondria. These morphological

changes would account for reduced oxidative phosphorylation reported in

isolated liver mitochondria of Mn-deficient mice and rats (Bell and

Hurley, 1973). The requirement of Mn as a cofactor for in vitro oxida-

tive phosphorylation was demonstrated in 1954 by Lindberg and Ernster.

Manganese has long been associated with arginase, an enzyme that

catalyzes the conversion of arginine to urea in mammals, although

arginase cannot be classified as a metalloenzyme. Activation of









arginase was suggested by Boyer et al. (1942) and Wachtel et al. (1943)
since Mn-deficiency in rats resulted in a decreased activity of this

enzyme. Arginase activity has subsequently been shown to depend heavily

on Mn (Hirsch-Kolb and Greenberg, 1968). Significance of Mn in the func-

tion of a diamine oxidase isolated from human placental tissue (Crabbe

et al., 1976) requires further elucidation. Bell and Hurley (1974)

reported reduced cytochrome oxidase and choline esterase activities,

and lower concentration of choline lipids in cephalic epidermis of Mn-

deficient fetal mice. Farnesyl pyrophosphate synthetase, isolated from

pig liver and required for the production of squalene, is known to re-

quire Mn for activity (Benedict et al., 1965). It is apparent that Mn

is involved in a wide range of enzyme activities in a variety of tissues,

and that mitochondrial structures and function are particularly affected

by Mn deficiency. This would account for the high concentration of Mn

in mitochondria as first reported by Maynard and Cotzias in 1955.


Manganese and Reproduction

The earliest experiments establishing the essentiality of Mn in

the diet of rats and mice demonstrated defective ovarian function and

testicular degeneration (Kemmerer et al., 1931; Orent and McCollum,

1931). Skinner et al. (1932) reported slower sexual maturation in female

rats on all-milk diets compared with those on the same diet with 10 ppm

added Mn. Boyer et al. (1942) observed absent or irregular estrus with
a delay in the opening of the vaginal orifice in female rats fed a low

Mn diet. Testicular degeneration, absence of libido, and sterility

associated with seminal tubule degeneration, reduced spermatogenesis,









and accumulation of degenerating cells in the epididymus were observed

in male rats and rabbits (Boyer et al., 1942; Smith et al., 1944).

Apgar (1968) recognized three stages of Mn deficiency in the female

rat depending on degree of deficiency. In the least severe stage the

animals gave birth to viable young, some or all of which had congenital

malformations expressed as ataxia. The second, more severe stage re-

sulted in stillbirths and neonatal deaths. In the acute stage of de-

ficiency estrus cycles were absent or irregular, and the animals would

not mate or were sterile. Omission of Mn from the diet of gestating

guinea pigs increased the proportion of young born dead or premature and

reduced litter size (Everson et al., 1959).

Manganese deficiency in ruminants has resulted in delayed, silent,

or absent estrus; reduced conception rates; delayed ovulation; ovarian

and testicular degeneration; increased embryonic mortality and abor-

tions; impaired spermatogenesis; reduced birth weights (Hidiroglou,

1979a,b); and abnormal development of fetal epiphyseal cartilage

(Hidiroglou and Knipfel, 1981). Bentley and Phillips (1951) reported

that heifers receiving only 10 ppm Mn were slower to exhibit first estrus,

slower to conceive upon breeding, and gave birth to a higher percentage

of calves with leg deformities than did control heifers raised on diets

with 30 ppm Mn. Delayed ovulation was associated with Mn deficiency in

cattle (Hignett, 1941; Rojas, 1965). Wilson (1966) reported failure to

come into heat and infertility associated with one or both ovaries being

subnormal in size. In the United Kingdom, Munro (1957) reported that

conception rates in some British cattle increased from 48 to 72% follow-

ing Mn supplementation. Results produced in cattle were duplicated to








varying degrees in goats (Anke et al., 1973a) and ewes (Egan, 1972;

Hidiroglou et al., 1978).

Low dietary Mn in poultry was linked to nutritional chondrodystrophy

in chick embryos that resulted in gross skeletal malformations and embryo

mortality (Lyons and Insko, 1937). Gallup and Norris (1939b) observed

low egg production and a high incidence of embryo mortality when layers

were fed a low Mn diet while Gutowska and Parkhurst (1942) found reduced

shell thickness, Ca, and breaking strength and increased egg shell de-

formation with low dietary Mn (Hill and Mathers, 1968; Panic et al.,

1978; Maurice and Whisenhunt, 1980; Whisenhunt and Maurice, 1981).

Studies of Longstaff and Hill (1971, 1972) on the mucopolysaccharide

content of the egg shell found low dietary Mn reduced uronic acid con-

centration thereby affecting egg shell strength.

Reproductive anomalies have also been reported in swine. Johnson

(1943) reported that sows fed low-Mn diets gave birth to dead or weak

pigs with high mortality, exhibited fetal resorption, and lacked normal

udder development and milk production. Grummer et al. (1950) duplicated

these findings and extended them to include irregular estrual cycles,

complete absence of estrus in some animals and absence of marked signs

of estrus in others, birth of small, weak pigs which could neither stand

nor walk, poor udder development and virtually no milk production when

sows were fed .5 ppm Mn. Manganese was reduced in milk, colostrum, and

body tissues due to low dietary Mn.

The precise biochemical lesions responsible for reproduction ab-

normalities are yet to be elucidated. On the basis of studies of Mn

distribution among tissues of the reproductive tract of normal and

anestrus ewes, it has been suggested that Mn has a possible role in the








normal metabolism and/or activity of the corpus luteum (Hidiroglou,

1975; Hidiroglou and Shearer, 1976). Hidiroglou et al. (1978) postu-

lated that since Mn is an activator of many enzyme systems, a lack of

dietary Mn might inhibit the active synthesis of material required for

complete fetal development and normal implantation. Doisy (1974) sug-

gested that lack of Mn inhibited the synthesis of cholesterol and sub-

sequent synthesis of sex hormones and possibly other steroids, thus

affecting fertility. This could be mediated through the effect of Mn on

activity of farnesyl pyrophosphatase synthetase during synthesis of

squalene (Benedict et al., 1965).


Carbohydrate and Lipid Metabolism

The defects of Mn deficiency as an expression of glycosyltrans-

ferases in carbohydrate metabolism have been discussed. Other defects

in carbohydrate metabolism have been observed in Mn deficient animals.

Everson and Shrader (1968) reported reduced glucose tolerance in Mn-

deficient guinea pigs. These animals showed decreased utilization of

glucose and exhibited a diabetic-like glucose curve in response to glu-

cose loading either orally or intravenously. Deficient animals were then

supplemented for 2 months and showed a normal response. The same workers

(Shrader and Everson, 1968) reported a high incidence of stillbirths and

poor viability of neonatal guinea pigs born to females maintained on Mn-

deficient diets throughout gestation. Necropsy revealed gross abnormali-

ties in the pancreas such as aplasia or marked hypoplasia of all cellular

components. Islet population was reduced where hypoplasia occurred but

islet size increased. Islets contained fewer and less granulated beta









cells. Young adult Mn-deficient guinea pigs also had decreased numbers

of pancreatic islets which enlarged in size, contained less intensely
granulated beta cells and increased number of alpha cells. Following

supplementation for 2 months, increased islet numbers were found which

contained more beta cells and increased granulation than deficient

animals.

The mechanism of glucose utilization with respect to Mn deficiency

is not known but may be related to the reduced mucopolysaccharide syn-

thesis that occurs in such animals. It has been suggested that Mn may

be involved in insulin formation or utilization (Underwood, 1977).

Insulin may regulate utilization of glucose in the synthesis of muco-

polysaccharides. Everson (1968) speculated that lower urinary myoinosi-

tol content in newborn guinea pigs might be related to the impaired

carbohydrate metabolism associated with skeletal defects.

The role the Mn metalloenzyme, pyruvate carboxylase, in the carboxy-

lation of pyruvate to oxaloacetate in the tricarboxlic acid cycle

(Mildvan et al., 1966) was discussed. There was also evidence of a role

of Mnin gluconeogenesis. Friedman and Rasmussen (1970) provided in vitro

confirmation of this with studies using isolated perfused rat liver.

Doisy (1974) proposed another relationship between Mn and carbo-

hydrate metabolism. Evidence of a Mn effect on the ability to elevate

clotting proteins in response to vitamin K was found in a patient with

coincident deficiencies of vitamin K and Mn. Hypocholesterolemia was

also observed in this man. The effect on blood clotting was confirmed

in chicks and it was suggested that Mn might play a role in the conver-

sion of preprothrombin to prothrombin, a glycoprotein whose synthesis may

be influenced by the effect of Mn on glycosyltransferases.








The reduced cholesterol observed in the human patient described

above appeared to confirm evidence of Curran (1954) who markedly in-

creased cholesterol synthesis in vitro by the addition of Mn to rat liver.

Manganese was first shown to be a cofactor for the conversion of mevalonic

acid to squalene (Amdur et al., 1957) which could affect subsequent

cholesterol synthesis. However, recent evidence with chicks, hens

(Klimis-Tavantzis et al., 1983a), normal rats, and genetically hyper-

cholesterolemic rats (Klimis-Tavantzis et al., 1983b) found no signifi-

cant alterations in cholesterol or lipid metabolism although Mn-deficient

rats were found to have reduced low density lipoproteins and the normal-

type rats had reduced hepatic fatty acid synthesis.
These recent studies contrast with the dramatic lipotropic effect

of Mn reported in swine (Plumlee et al., 1956), mice (Bell and Hurley,

1973), and rat (Amdur et al., 1946) studies which found increased fat de-

posits in Mn-deficient animals. Choline supplements had a similar effect

upon lipid metabolism and changes in ultrastructure of the liver and may

be linked in a common pathway affecting mitochondrial structures and

cellular membranes (Evans et al., 1943; Amdur et al., 1946; Bell and

Hurley, 1973). While it is apparent that Mn is important for normal

carbohydrate metabolism and required by both the pancreas and liver,

there is much that remains to be learned about the relationship of Mn

to carbohydrate and lipid metabolism.


Manganese Metabolism

Homeostasis

Mineral homeostasis is the combination of factors working in the

body which maintain tissue mineral levels within relatively narrow margins





-20-


even when animals consume a considerably wider range of the mineral in

the diet. This is an important biological survival mechanism considering

the tremendous difference in Mn concentration found in various animal

feeds (Shroeder et al., 1966; Adams, 1975). These factors include en-

hanced absorption, altered tissue retention, and reduced excretion when

dietary levels are low and conversely, increased excretion with reduced

absorption and altered tissue turnover when dietary levels are high.

The relative importance of absorption, excretion, and tissue turnover is

dependent upon the particular element.

This concept is generalized in figure 1 which indicates tissue

mineral concentration with respect to dietary level, with and without

the mechanisms of homeostasis in operation. When dietary levels are low

there is enhanced utilization. This effect is due to increased absorp-

tion (Settle et al., 1969; Suso and Edwards, 1969; Abrams et al., 1976),

decreased excretion (Britton and Cotzias, 1966; Papavasiliou et al.,

1966; Bertinchamps et al., 1966) and altered tissue retention and turn-

over (Britton and Cotzias, 1966; Lassiter et al., 1974).

Present evidence indicates biliary excretion into the gastro-

intestinal tract (Britton and Cotzias, 1966; Papavasiliou et al., 1966)

and endogenous excretion (Miller et al., 1972, 1973; Carter et al.,

1974; Abrams et al., 1977) are of primary importance in the homeostasis

of Mn and that intravenously administered Mn is metabolized quite dif-

ferently from that of absorbed Mn. Absorption also plays a smaller but

significant role in homeostasis (Lassiter et al., 1974; Abrams et al.,

1977). The various mechanisms appear to be interdependent and together

they constitute efficient homeostasis. Evidence indicated that Mn

excretion was almost exclusively via the feces and significant excretion





























INTAKE LEVEL


FIGURE 1. Influence of homeostasis on tissue mineral concentration with respect to
dietary intake level








in the urine occurred only in the presence of chelates, such as EDTA

(Maynard and Fink, 1956; Cotzias, 1958).

Bile was regarded as the principal route of Mn excretion in mammals

(Greenberg et al., 1943; Thomas, 1970) and enterohepatic circulation of

bile has been considered the principal regulatory mechanism of Mn

homeostasis (Papavasiliou et al., 1966; Bertinchamps et al., 1966).

It was also demonstrated that Mn could be excreted via the duodenum and

jejunum (Koshida et al., 1963; Kato, 1963; Bertinchamps et al., 1966) and

pancreas (Burnett et al., 1952). These were considered auxiliary mechan-

isms which participated in excretion primarily under conditions of over-

loading (Papavasiliou et al., 1966). Miller et al. (1972) suggested that

much of the Mn absorbed goes back into the intestinal contents soon after

uptake. It was known that endogenous excretion of Mn was sensitive to

levels of dietary Mn (Cotzias and Greenough, 1958; Bertinchamps et al.,

1966; Watson et al., 1973). In addition, it became clear that intra-

venously administered Mn was metabolized quite differently from that of

absorbed Mn (Watson et al., 1973; Miller, 1975). Newer evidence (Miller

et al., 1973; Carter et al., 1974; Abrams et al., 1976, 1977) supported

the hypothesis that rapid endogenous excretion was perhaps the most

important variable in Mn homeostasis. This is consistent with the idea

put forth by Cotzias (1962) that the gastrointestinal contents may be

considered as a Mn pool which is in more rapid equilibrium with the

tissues than it is with the outside world.

At high dietary levels (figure 1), homeostasis is overcome and

toxicity ensues. The capacity of the liver to extract and excrete Mn

can be exceeded which may lead to a breakdown in excretory functions

resulting in higher plasma Mn concentration and increased tissue Mn









accumulation (Hall et al., 1981). Mertz (1983) stated that redistribu-

tion of excessive amounts of elements to "dumping sites" within the

organism in relatively inert chemical forms which are not in equilibrium

with the metabolically active pool of the element (e.g., bone storage)

is important in protecting against high dietary intakes. Homeostasis

of Mn is highly efficient and Miller (1973) suggested that the range

between a deficient and toxic mineral level is determined in large by

the effectiveness of homeostatic control mechanisms.


Absorption and Excretion

Absorption of Mn is quite low. The early studies of Greenberg et

al. (1943) indicated absorption of only 3 to 4% in rats. More recently,

absorption in ruminants was determined to be approximately 1% (Sansom

et al., 1976; Abrams et al., 1977) or less (Sansom et al., 1978) in

adult animals. Much higher absorption rates have been observed in milk-

fed cattle (Carter et al., 1974) and rats (King et al., 1980; Rehnburg

et al., 1980). There was also an age effect on Mn absorption. Decreases

in Mn absorption within the first few days of life have been reported

for the rat (Mena, 1974; Cotzias et al., 1976; Cahill et al., 1980),

mouse (Miller et al., 1975), calves (Howes and Dyer, 1971), chicks

(Mathers and Hill, 1967), and humans (Shroeder et al., 1966; Mena, 1981).

Kirchgessner et al. (1981) recently provided in vitro evidence of this

effect by measuring intestinal uptake of radioactively labeled Mn in
everted jejunal sacs. Le Feure and Joel (1977) suggested that pinocyto-

tic activity of the intestinal epithelial cells in the immature digestive

tract allowed particulate material to be absorbed, dissolved, and trans-

ported to the portal blood.









The mechanism of Mn absorption is not precisely known. Thomson

et al. (1971) found Mn to be absorbed equally well throughout the length

of the small intestine while Grace (1975) reported that net secretion of

Mn occurs in the small intestine and net absorption in the cecum and

colon. Evidence suggesting that some Mn absorption is by an active

process has been reported, based on in vitro studies that showed a

reduction in uptake of 52Mn through inverted duodenal sacs under anaerobic

conditions (Cikrt, 1970). Ingested Mn+2 is converted to Mn+3 in the

alkaline duodenal medium. Absorption of Mn+3 is dependent upon the

intestinal concentration of Mn and the level and form of the Mn and

organic chelates in the body (Venugopal and Luckey, 1978).

Some of the earliest studies (Skinner et al., 1931; von Oettingen,

1935; Greenberg et al., 1943) reported that Mn is excreted primarily in

the feces. This has been supported by other studies which indicated

only minor excretion (<1%) of Mn occurred in the urine under normal

circumstances, even with high dietary levels (Watson et al., 1973).

Mechanisms of gastrointestinal excretion were discussed in relation

to homeostasis of Mn.


Blood Clearance, Tissue Retention, and Turnover

Sansom and coworkers (1976) presented evidence that Mn ions ab-

sorbed into the portal circulation may remain free or bound to a2-

macroglobulin before traversing the liver, where they are removed almost

completely, although some of the protein bound Mn may enter the systemic

circulation and become bound to a 01-globulin carrier protein, probably

not different from transferrin. Borg and Cotzias (1958a,b) and Cotzias









(1963) reported that within 10 minutes of intravenous injection of 54Mn

and 56Mn, only 1% of the dose remained in the plasma and after 70 minutes

radioactivity was barely detectable in plasma. This rapid clearance

occurred in three phases. The first and most rapid of these is identical

with the clearance rate of other small ions suggesting normal trans-

capillary movement. The second was identified with the entrance of Mn

into the mitochondria of the tissues. The third and slowest component

could be associated with the rate of nuclear accumulation of the metal.

The rapidity with which the liver mitochondrial pool comes into equilib-

rium with the plasma pool suggested that this represents a dynamic,

mobile pool of tissue Mn. Hansard (1972) noted that the kinetic pattern

for blood clearance and for liver Mn uptake appeared to be identical

indicating the two Mn pools rapidly attain equilibrium.

Tissue analyses of several species have indicated similar Mn con-

centration in the tissues of rats (Skinner et al., 1931), rabbits (Fore

and Morton, 1952), cattle (Bentley and Phillips, 1951; Standish et al.,

1969; Ivan and Grieve, 1975), sheep (Standish and Ammerman, 1971; Watson

et al., 1973; Doyle and Pfander, 1975), chickens (Mathers and Hill,

1968; Kienholz et al., 1974), swine (Leibholz et al., 1962; Svajgr et

al., 1969; Gamble et al., 1971), and humans (Bruckmann and Zondek, 1939;

Shroeder et al., 1966). Manganese concentration ranged from approxi-

mately .5 to 12 ppm (dry matter basis) and was more concentrated in bone,
pituitary, liver, kidney, and pancreas while considerably less was found

in muscle, heart, lung, brain, spleen, and other tissues. For a more

detailed account of Mn in animal tissues, the reader is referred to

Shroeder (1966) and Underwood (1977).









Tissue turnover of Mn is very dynamic compared to other trace ele-

ments such as Zn (Miller, 1973). The turnover of 54Mn is relatively

fast in liver, gallbladder, spleen, small intestine, and some other

visceral organs, but substantially slower in muscle, bone, and skin.

Several studies have shown that tissue retention time decreased as the

dietary load of Mn increased (Papavasiliou et al., 1966; Britton and

Cotzias, 1966; Miller et al., 1973; Watson et al., 1973). In mice, for

example, a 10-fold increase in dietary Mn reduced excretion time of a

tracer dose of 54Mn by approximately one-half (Britton and Cotzias,

1966).

Evidence suggests that Mn metabolism may be under regulatory control

via hormones. An increase by a factor of two in the Mn concentration in

blood plasma of pullets with the onset of egg-laying (Hill, 1974) has

been associated with an increase in estrogen activity (Underwood, 1977).

Adrenal glucocorticosteroids administered to mice markedly altered 54Mn

distribution from the liver to other tissues of the carcass (Hughes and

Cotzias, 1961). Similar redistribution of Mn was obtained when ACTH was

administered to stimulate the animals' own adrenal cortices (Cotzias,

1958; Hughes et al., 1966). Stability of tissue level could not be

ascribed solely to the function of the adrenal cortical hormones because

adrenalectomy did not alter the Mn concentration in tissues, except in

animals receiving high-Mn intakes (Hughes et al., 1966).


Manganese and Mineral Interactions

It has become evident in recent years that a number of trace

element deficiencies encountered under practical conditions may result









not from a low intake of the element but from the presence of condi-

tioning factors in the diet (Bremner and Davies, 1973). Mineral ele-

ments which have similar electronic structure may act interchangeably

and sometimes antagonistically (Thomas, 1970; Matrone, 1974). This
idea is consistent with the observation that several metalloenzymes and

metal-enzyme complexes are nonspecific in the element required for

activation, and that Mn has been found to be incorporated into the heme

molecule in vivo and in vitro (Borg and Cotzias, 1958a) which is indis-

tinguishable from that of Fe porphoryn with respect to synthesis and

turnover.


Iron

A Mn-Fe interaction has been recognized for over 45 years (Wilgus

et al., 1937a). Perla and coworkers (1939) reported that high levels

of Mn reduced Fe stores in rats while Wilgus and Patton (1939) demon-

strated that ferric citrate added to a chick diet increased the incidence

of perosis and that ferric hydroxide removed Mn from solution in vitro.

Subsequently, Fe was shown to have a mutually antagonistic effect with

Mn manifested by reduced Mn absorption and increased Mn deficiency

lesions when excess Fe is present in the diet or reduced Fe absorption

and hemoglobin synthesis in the case of excess Mn. Excess dietary Fe

has produced conflicting results in regard to Mn metabolism (Wilgus et

al., 1937a). Standish et al. (1971) reported reduced Mn in the heart

and kidney of steers fed 1000 ppm Fe as reagent grade FeS04 but no Fe

effect on tissue Mn concentration in other studies using 1600 ppm (as

FeSO4) in steers (Standish et al., 1969) or sheep (as ferrous sulfate or

ferric citrate) (Standish and Ammerman, 1971).









More definitive evidence of a Mn-Fe interaction has been obtained

when high Mn levels (1000 to 4000 ppm) were added to the diets of

animals, particularly in the presence of an Fe deficiency or anemia.

A relationship between Mn, Fe, and hemoglobin formation was demonstrated

in pigs, rabbits (Matrone et al., 1959), lambs (Hartman et al., 1955),

rats (Baxter et al., 1965), and chicks (Southern and Baker, 1983a).

Hemoglobin regeneration was depressed as was Fe concentration in several

tissues including serum, liver, kidney, and spleen when dietary Mn was

increased. Hemoglobin synthesis was reduced at lower dietary levels than

those that reduced soft tissue Fe. The depressing effects of Mn could

be overcome by increasing dietary Fe. Robinson et al. (1961) reported

that high dietary Mn increased Fe excretion in calves but had no effect

on hemoglobin. Similarly, other groups found that Mn did not always

affect hemoglobin formation (Leibholz et al., 1962; Cunningham et al.,

1966). Depression of Fe stores was more consistently observed when high

dietary Mn was fed (Watson et al., 1973; Grace, 1975; Ivan and Hidiroglou,

1980; Southern and Baker, 1983a).

Neathery et al. (1982) reported evidence in calves suggested inter-

action of Mn and Fe was at the absorption site. Studies with rats indi-

cated that the effect of high Mn was greater on absorption than utiliza-

tion (Hansard et al., 1960; Diez-Ewaldetal., 1968; Forth, 1970). This con-

clusion was based on evidence that in Fe deficiency, not only Fe but Mn

and other chemically related metals are absorbed more efficiently. Iron

is preferentially absorbed by a system involving a two-step kinetic

process, but Mn in high concentrations competitively inhibited Fe

absorption (Forth, 1970; Thomson, 1971).









Calcium and Phosphorus

Manganese also interacts with Ca and P, primarily in the gastro-

intestinal tract. Hammond (1936) observed an effect of inorganic phos-

phate on increasing the incidence of perosis. Other evidence indicated

this causative effect was not due to P alone, but rather to high Ca in

the diet (Hunter et al., 1931; Payne et al., 1932; Wilgus et al., 1937b;

Caskey and Norris, 1938). Several studies have shown that high P, low

Ca diets did not aggravate perosis (Schaible et al., 1933; Clifcorn et

al., 1938) which led Wilgus and Patton (1939) to suggest that insoluble

calcium phosphate can remove manganous ions from solution by adsorbance.

This effect was confirmed by Schaible and Bandemer (1942). Effects of

Ca and P on Mn metabolism were also noted for cattle (Hawkins et al.,

1955; Dyer and Rojas, 1965), sheep (Suttle and Field, 1970), and pigs

(Kayongo-Male et al., 1974b). Recently, Summers et al. (1978) and

Cook (1982) were unable to duplicate lesions reported previously using

high dietary Ca. They reported dietary P levels of approximately .75%.

Cook and Crenshaw (1983) suggested that since in all papers reporting a

Mn deficiency lesion except one (Combs et al., 1942), chicks were fed

diets that contained .9% P or greater, then P was also important in

affecting Mn availability. This supported the conclusion of Schaible

and Bandemer (1942) that phosphates were more effective at adsorbing Mn

than carbonates, based on in vitro studies showing that bone meal and
calcium phosphate adsorbed Mn more efficiently than calcium carbonate.

Manganese interacts with Ca and P in other ways besides adsorption
which are less clear. Hidiriglou et al. (1978) reported reduced muscle

and kidney Ca in Mn supplemented ewes. However, in day-old lambs born









to these ewes, kidney and heart had higher Ca concentration. Robinson

et al. (1961) reported higher P excretion in calves supplemented with

600 ppm Mn while Anke et al. (1973b) found higher P in the heart, kidney,

liver, and skeletal muscle in kids of dams fed 1.9 ppm Mn compared to

those from dams receiving 90 ppm Mn. Injected Mn increased serum P and

reduced serum Ca in rats (Baxter et al., 1965). More recently, dietary

P was shown to reduce Mn in heart of lambs (Rosa et al., 1982) and in
the muscle of sheep (Valdivia et al., 1982). High dietary P increased

Mn excretion and high dietary Mn (>1000 ppm) decreased Ca and P balances

(Reid et al., 1947; Gallup et al., 1952; Hawkins et al., 1955; Cotzias,

1958; Meghal and Nath, 1964).


Copper and Zinc

It is clear from several studies that Mn interacts with Cu and Zn
in a way, as yet, not understood. The mode of action has not been de-

lineated for either element, and some of the evidence has been contra-

dictory. An interaction with Cu was described by Gubler et al. in 1954

who observed several responses when rats were fed a basal diet supple-

mented with 4% manganous chloride or .1% copper sulfate or a combination

of both. The Mn supplement either with or without Cu produced a micro-

cytic, hypochromic anemia. There was a slight reduction in Fe; however,

the anemia was morphologically similar to anemia associated with Cu

deficiency. Manganese alone increased Cu in plasma and brain, decreased

Cu in kidney, and had no effect on liver Cu. Urinary excretion of Cu
was decreased and these workers concluded that Mn caused a redistribution

of tissue Cu in these animals while total body Cu was not changed. While








the high Mn diet slightly reduced total Cu in rats, the high Cu treat-

ment increased total body Cu by a factor of three. An unexpected

synergistic effect occurred when high Mn and Cu were in the diet which

doubled total body Cu relative to the high Cu treatment.

The additive response of supplemental Cu with Mn was also reported

by Ivan and Grieve (1975) in calves, who also noted decreased urinary

excretion of Cu in response to dietary Mn. These workers reported that

dietary Zn or Cu had no effect on Mn. Dietary Mn increased Cu in the

liver of cattle (Gallup et al., 1952; Ivan and Grieve, 1975) and sheep

(Watson et al., 1973). Contrary to these reports Ivan and Hidiroglou

(1980) found reduced liver Cu in sheep fed up to 3000 ppm Mn and Anke

et al. (1973b) reported that 90 ppm Mn decreased Cu in heart, skeletal

muscle, and liver of goats compared to animals fed a Mn deficient diet.

Kienholz and coworkers (1974) reported no effect on tissue Mn of chickens

fed 500 ppm Cu with or without 2000 ppm Mo. Ivan and Grieve (1976)

reported that dietary Cu decreased the net absorption of Mn up to the

omasum and that dietary Mn decreased total net absorption of Cu in calves.

They also found that Zn increased net absorption of Mn in some sections

of the gastrointestinal tract although overall there was no change.
Evidence of a Mn-Zn interaction has also been conflicting. Ivan

and Grieve (1975) found that dietary Mn increased Zn in liver, heart,

and kidney of calves. Similarly, Zn was increased in heart, kidney,

and muscle of goats by Mn supplementation compared to Mn deficient goats

(Anke et al., 1973b). Grace (1973) reported that Mn supplemented sheep

had higher Zn concentration in heart while Hidiroglou et al. (1978)

found increased Zn in spleen and decreased Zn in heart of ewes supple-
mented with Mn. High dietary Mn (4000 ppm) reduced Zn in liver of sheep









(Watson et al., 1973) while 2000 ppm Zn was shown to reduce Mn in
Japanese quail (Hamilton et al., 1979). Ott et al. (1966) fed 3500 ppm

Zn to lambs and reported no change in Mn concentration in liver.

These results indicate that more research is necessary to more

fully understand the interaction of Mn with the metabolism of Cu and

Zn.


Magnesium and Other Elements

The interchangeability of Mn and Mg in many enzyme systems in vitro

and in vivo was discussed. Biologically, however, the intact mammalian

body discriminates quite efficiently between the transition element Mn

and the alkaline earth element Mg. Blakemore and associates (1937) re-

lated high forage Mn to incidence of lactation tetany, an observation

yet to be substantiated. Hawkins et al. (1955) noted the depressing

effect of high levels of Mn on serum Mg in calves. Fain et al. (1952)

reported that 100 ppm supplemental Mn reduced serum Mg, but this effect

was not found at 75, 150, or 200 ppm. A different effect was observed

by Baxter et al. (1965) who reported that rats administered acute Mn

injections (15 mg/100 g body weight of manganous chloride) increased in

serum Mg concentration.

More recently Hidiroglou et al. (1978) fed low Mn (8 ppm) and Mn

supplemented (68 ppm) diets to ewes. Subsequent tissue analysis of these
animals and their day-old progeny revealed numerically lower Mg concen-

trations in pancreas, adrenal gland, liver, lung, and spleen of the

group of Mn supplemented ewes, but the difference was significant only

for spleen. In lambs from the supplemented group, Mg was lower in heart









and also numerically lower in liver, lung, muscle, and kidney. These

workers speculated that because of the similarity in chemistry between

Mg+2 and Mn+2 and that they can replace each other in several biochemical

systems, it is possible that dietary Mg was utilized to a greater extent

by the body of animals on low Mn diets.

Cotzias (1961) discussed the differentiation of the mammalian body

between Mg and Mn. He maintained that this dichotomy may be explained

on the basis that Mn assumed a valence of +3 in vivo while Mg does not.

In vitro studies have primarily been conducted with both elements in the

divalent state.

Less profound mineral interactions have been observed that merit

mentioning. Hidiroglou et al. (1977) found that addition of S to a

corn silage diet supplemented with urea reduced serum Mn levels in sheep

compared to the serum of sheep on low S diets. Valdivia and coworkers

(1982) reported that dietary Al reduced Mn in liver and increased Mn

concentration in bone. Manganese can reduce the toxicity of Pb (Mylroie

and Patterson, 1979) in a manner not yet established. These workers

found that Pb ingestion reduced tissue Mn levels. Pearl et al. (1983)

studied Pb accumulation and depletion in sheep and compared tissue

mineral levels of pre-treatment controls versus accumulation, and

depletion phases. Manganese in the brain was increased during the

accumulation and depletion phases while muscle Mn was greater during

the depletion period. Lomba et al. (1975) reported that K intake can

affect the dietary Mn requirement of cattle and that animals which re-

quire larger amounts of K should also have their Mn intake increased.
The significance of these interactions remains to be determined.

It is evident that Mn interacts with other elements, most notably by









competitive inhibition, by initiating redistribution of these elements,

and/or by influencing tissue retention and excretion rates.


Manganese Requirements


The Mn content of feeds is highly variable. The reader is referred

to Peterson and Skinner (1931), Shroeder et al. (1966), and National

Academy of Science (1973) for detailed information on the Mn content of

various feedstuffs. In general, most diets provide adequate Mn to meet

requirements. The most important exception occurs when corn, sorghum,

or corn silage comprises a major portion of the diet. These feeds are

substantially lower in Mn content than other feedstuffs. Unless sup-

plemented, diets based largely upon these feeds, such as modern poultry

diets containing high levels of corn, will be inadequate in Mn (Underwood,

1981). The requirements of animals for Mn are also variable with avian

species generally higher than mammalian species, and dependent on a

number of factors. These factors include the criteria of adequacy em-

ployed, species or genetic strain, age, level of production, chemical

form and bioavailability, and composition of the diet.

The dietary requirements for growth and maintenance have been

found to be lower than requirements for reproduction, production, or

other stress factors, in several species (Underwood, 1977). Breed and

strain differences for requirements of Mn for prevention of perosis

(Gallup and Norris, 1939a) and for egg production (Golding et al., 1940)

were recognized over 40 years ago. Similar observations were reported

for mice (Hurley and Bell, 1974). Age effects can be related to stage

of growth, reproduction, or production. Bioavailability of Mn is









determined by its chemical form, solubility, and presence of antagonists

in the feed or contaminants of inorganic Mn sources. Soybean meal pro-

tein was shown to reduce the availability of Mn for the chick (Davis

et al., 1962) and Davies and Nightingale (1975) demonstrated that this

reduction is related to the high phytate content of soybean meal.

Laboratory species such as mice, rats, rabbits, and guinea pigs are

unable to grow normally on diets containing less than .3 ppm (Daniels

and Everson, 1935; Wachtel et al., 1943; Smith et al., 1944). Smith

and Ellis (1947) found 300 ag/day satisfactory for the growth of rabbits

but the same amount was suboptimal for rats. A level of 40 to 45 ppm

was adequate for growth and normal reproduction in mice and rats

(Holtkamp and Hill, 1950; Hurley et al., 1960; Bell and Hurley, 1973).

The minimum dietary requirement for ruminants has been difficult

to assess with precision. Hawkins et al. (1955) suggested that the

minimum requirement for the growth of young calves was not more than

1 ppm. Bentley and Phillips (1951) reported that 10 ppm dietary Mn was

adequate for growth of heifers. They concluded that growth and normal

reproduction were maintained with 20 ppm; however, Dyer et al. (1964)
reported that 61 ppm was the minimum level which resulted in normal

calves, and they found numerous deformities in calves from dams fed

21 ppm. There were no definitive studies for sheep and goats. Anke

et al. (1973a) found impaired reproduction at dietary levels of 20 ppm orless

forgoats. It is likely that the requirements of sheep and goats are very
similar to cattle. The National Research Council suggested a require-

ment of 20 to 40 ppm for sheep (1975); 1 to 10 ppm for growing and
finishing beef cattle and 20 ppm for dry, pregnant cows (1976); and

40 ppm for all classes of dairy cattle (1978).









The National Research Council (1977) recommended 55 ppm as the

minimum dietary requirement for the growth of chicks. This information

was based largely on the studies of Gallup and Norris (1939a)using diets

which were relatively high in Ca and P. It is likely that 40 ppm is

adequate (Underwood, 1977); however, a margin should be included when

Ca and P are greater than 1.2 and .9%, respectively. A level of 25 ppm

was recommended for layers and 33 ppm for breeding hens (National Re-

search Council, 1977). Several studies indicated that maximum shell

strength was obtained at levels greater than 50 ppm (Longstaff and Hill,

1971, 1972; Maurice and Whisenhunt, 1980; Whisenhunt and Maurice, 1981).

Cook and Crenshaw (1983) suggested that 75 to 80 ppm is optimal for

laying hens. Levels for turkeys appeared to be somewhat higher and are

dependent on dietary Ca and P (Atkinson et al., 1967).

Manganese requirement of swine is lower than other classes of live-

stock. Plumlee et al. (1956) reported no evidence of deficiency when

pigs were fed 3.5 ppm Mn. Johnson (1943, 1944) suggested that the re-

quirement was probably much lower, approximately .5 ppm for maximum

growth. Reproduction was not normal for pigs receiving .5 to 1.5 ppm;

however, Grummer et al. (1950) found only slightly improved reproductive

performance when a corn diet containing 12 ppm was supplemented with

additional Mn. Several studies have indicated that the dietary Mn re-

quirement of pigs for optimum growth and reproduction is well below levels

found in corn-soybean meal diets typically fed to pigs (Liebholz et al.,

1962; Svajgr et al., 1969; Omole and Bowland, 1974). The National

Research Council (1979) recommended 2 to 4 ppm Mn for growing-finishing

swine fed ad libitum and 10 ppm for breeding swine.









Manganese Bioavailability

In the study of trace element metabolism, it is recognized that the

total level of a mineral in a particular compound or feed must be quali-

fied by a factor which indicates to what degree the nutrient is utilized

when consumed by animals. Concentrations of trace elements in feeds

and supplements are of limited use if the amount utilized is obscure.

The choice of an inorganic source of a trace element is dependent on

handling and mixing properties, presence of toxic contaminants, dependa-

bility and uniformity of supply, and relative cost per unit available

element (Ammerman and Miller, 1972). Thus, the relative biological

availability of Mn sources is a major consideration in the choice of a

suitable source of the element.

In general, the bioavailability of an element is determined rela-

tive to its functional availability from a standard source. These

studies are conducted using dietary levels which are below minimum

requirements. The relative value of different sources in preventing

the appearance of deficiency lesions may then be determined. Use of a

standard allows expression of bioavailability in terms of relative

biological availability or value as described for Fe by Fritz et al.

(1970). Research to determine the relative biological availability of

Mn has been conducted primarily with poultry. This is due to the fact

that Mn deficiency is of practical nutritional concern with poultry and

has not been shown with certainty to occur in ruminants or swine. In

addition, effective measures of Mn availability have been difficult to

develop with some species, particularly ruminants (Watson, 1968).

Schaible et al. (1938) compared the effectiveness of several Mn
sources based upon reduction in incidence of perosis when 30 ppm Mn were





-38-


supplemented to a diet containing 11 ppm Mn. These workers found sul-

fate (MnSO4.2H20), chloride (MnCl2.2H20), carbonate (MnC03), dioxide

(Mn02), and permanganite (KMn04) equally effective in preventing the

occurrence of perosis. Additionally, the natural oxide ores manganite

(Mn203), pyrolusite (Mn02), psilomelane (Mn02?), and hausmannite (Mn304)

yielded results equivalent to those obtained from reagent grade sources,

while rhodocrosite (MnC03) and rhodonite (MnSi03) were considerably less

effective. They suggested that contaminants present in rhodocrosite

interfered with the utilization of Mn and that the silicate ore was in-

soluble. Gallup and Norris (1939a) found similar results using chemically

pure MnCl2*4H20, MnSO4.4H20, KMnO4, MnC03, and MnO2. Each of these

salts was equally effective in reducing perosis when added to a basal

diet containing 10 ppm at a rate of 40 ppm.

Bandemer and coworkers (1940) compared the natural carbonate ore

rhodocrosite to a precipitated carbonate (MnC03). Their findings sup-

ported earlier studies which indicated rhodocrosite was ineffective in

preventing perosis, even at levels of 125 ppm, while the precipitated

carbonate was completely effective when supplemented at 30 ppm. These

workers (Bandemer et al., 1940) also conducted experiments to determine

the effects of particle size, impurities, and processing of the ore.

They found that reducing particle size to a level that would remain

suspended in alcohol for 5 minutes was without effect in reducing perosis.
Roasting or dissolving rhodocrosite in HCI, as well as MnCO3 precipi-

tated with known impurities were effective in reducing perosis and

indicated that the low utilization of Mn from this ore was not due to

impurities as proposed earlier by Schaible et al. (1938).





-39-


Combs (1951) reported that the sulfate, chloride, carbonate,

dioxide, and potassium permanganate forms, as well as the ores, man-

ganite, pyrolusite, hausmannite, and hematite were satisfactory Mn

sources for preventing perosis in the chick. He found that an oxide

ore was 85% as effective as sulfate in preventing perosis when supple-

mented at levels of 15 or 25 ppm but was of equal value when added at

levels of 35 and 55 ppm. These results suggested that incidence of

perosis was not a highly sensitive measure of bioavailability and that

there might be real differences in availability of various sources shown

previously to be equal in value. Henning et al. (1967) measured uptake

of radioactive Mn in broilers from 54Mn02, 54MnSO 4, and 54MnC12. These
workers found that radioactive Mn as the chloride was incorporated into

the body at a higher rate than that from the other compounds.

More recent studies (Watson et al., 1970, 1971) found that leg

deformity score was a more sensitive criterion for availability than

body weight, bone ash, or bone Mn concentrations. It was apparent that

this method was able to discriminate availability more precisely than

prevention of perosis. These workers used several Mn sources supple-

mented to a basal diet containing 5 ppm Mn. These sources provided

10 ppm supplemental Mn and chicks were fed to 28 days of age. Leg

scores indicating relative degree of perosis were used as the criterion

of bioavailability. They reported that reagent grade sulfate (MnSO4.H20)

and carbonate (MnCO3) were equally available while several oxide ores

had varying degrees of availability. Determination of the components

of these ores by X-ray diffraction indicated that the more available
oxides were composed primarily of manganosite (MnO) and that availability

was influenced by the relative proportions of MnO to MnO2 and the








presence of mineral impurities. These workers reported low availability

of rhodocrosite similar to the findings of Schaible et al. (1938) and

Bandemer et al. (1940). They also reported a high correlation between

their in vivo studies and an in vitro determination of solubility in

neutral ammonium citrate. They concluded by suggesting that a more

sensitive bioassay might result if higher levels of added Mn were sup-

plied from test sources using bone Mn as the response criterion.

No definitive studies on bioavailability of Mn sources in ruminants

have been reported, although Mn as the sulfate or chloride were shown

to be effective sources for cattle (Bentley and Phillips, 1951; Dyer et

al., 1964; Rojas et al., 1965). Reagent grade MnSO4.H20, MnCO3, and

MnO were found to be equally available to weanling pigs when 10 ppm Mn

was added to a basal diet containing 16 ppm Mn (Kayongo-Male et al.,

1974). More recently, King et al. (1979) were not able to detect dif-

ferences in availability between reagent grade MnCO3 or the much more

soluble MnCl2 when fed to rats at low dietary levels (<3 ppm). No

systematic investigations have been conducted to determine the avail-

ability of Mn from feedstuffs. Erway and Purichia (1974) proposed that

formation of otoconia in the mouse ear might be a particularly sensitive

bioassay for Mn.
It is evident from the information presented that more precise

measures of relative biological availability of Mn are needed.


Manganese Toxicosis

Extensive reviews have summarized the effects of Mn toxicosis from

acute parenteral lethal doses in laboratory animals (Select Committee on





-41-


GRAS Substances, 1979), industrial Mn intoxication in man (National

Academy of Sciences, 1973), and excessive oral intakes in domestic

animals (National Research Council, 1980). The National Research Council

(1980) reported that feeding excess Mn (levels up to 1000 ppm) produced

detrimental results in only one of twenty-one experimental groups cover-

ing several species. The present review will discuss only effects of

high oral Mn. Manganese is among the least toxic of trace elements,

especially in the divalent form (Underwood, 1977). Due to poor absorp-

tion and variable excretion, acute oral toxicity studies have failed to

produce significant lethal effects. It was suggested (Select Committee

on GRAS Substances, 1979) that toxic evaluation of Mn in animal feeds is

not customarily based on lethality, but upon depression of appetite and

growth rates. However, effects on hemoglobin regeneration and mineral

interactions have been noted with high dietary levels.

Becker and McCollum (1938) reported no adverse effect in rats fed

MnCl2"H20 for extended periods at levels up to 4990 ppm. At levels

twice this high they observed slightly depressed growth, but no effect

on reproduction. Chornock and coworkers (1942) reported that rats fed

diets containing 1.73% Mn had increased loss of fecal Ca, a negative P

balance, retarded growth, and severe rickets. Rats fed massive doses

of Mn (4% manganous chloride) exhibited microcytic, hypochromic anemia,

reduced tissue Fe, and a change in tissue partition of Cu (Gubler et al.,

1954). High dietary Mn produced hypoplasia of the dental enamel in rats

(Wessinger and Weinmann, 1943). Moinuddin and Lee (1960) observed no

toxic effects of 3800 ppm Mn from MnSO4.H20 fed to rats for four days.

However, when 7586 ppm was fed for four weeks, several effects were noted

including decreased feed intake, hemoglobin, body weight, and serum P





-42-


and poorer feed conversion. Pigment in the incisor was reduced and water

consumption, urine volume, and RBC count were increased. Hansard et al.

(1960) reported depressed growth and Fe absorption at levels as low as

500 ppm in growing rats.

More recently, rats dosed with up to 20 pg/g body weight daily by

oral gavage showed no visible signs of toxicosis and exhibited normal

growth, reduced endogenous dopamine (Deskin et al., 1981a), and increased

serotonin (Deskin et al., 1981b) in the hypothalamus. Gray and Laskey

(1980) reported that 1050 ppm Mn retarded sexual development and lowered

reactive locomotor activity levels in mice exposed chronically to Mn304.

Carter et al. (1980) found decreased serum creatinine and increased

serum Ca and P in rats fed 1100 ppm Mn (as Mn304) from birth to 100 days.

When guinea pigs were fed 4.37 mg Mn/kg body weight by gavage (as

MnCl2) for 30 days, there was increased mortality, gastric and intestinal

mucosal necrosis, and altered enzyme activity (Chandra and Imam, 1973).

A single dose of MnSO4 (182 mg/kg body weight) by gavage produced no

deaths among hamsters (Fairchild et al., 1977). Young rabbits consuming

2.3 mg/d Mn in drinking water had slightly reduced growth rate (Umarji

et al., 1969). Those consuming 24.4 mg/d exhibited weight loss, anes-

thesia of the extremities, and transient paralysis.

The antagonistic effect of high Mn levels upon Fe metabolism and

hemoglobin regeneration was discussed in reference to Mn interactions.
Matrone et al. (1959) demonstrated that hemoglobin regeneration in

mature rabbits was reduced in animals fed 2000 ppm Mn, although in-

creasing Fe supplementation to 400 ppm alleviated this effect. Similar

Mn levels reduced hemoglobin formation in baby pigs. Dietary levels of

1250 and 2000 ppm resulted in reduced growth. These results confirmed









the findings reported by Hartman et al. (1955) using MnSO4*H20 in lambs.
These workers found depressed hemoglobin formation and tissue Fe con-

centration when anemic lambs were fed as little as 45 ppm Mn. They

suggested that Mn interferes with Fe at the absorption site and postu-

lated that excessive Mn antagonizes the enzyme systems that oxidize or

reduce Fe at the absorption site.
Watson and associates (1973) fed wether sheep a basal diet supple-

mented with 4000 ppm Mn from reagent grade MnCO3. They reported increased

Cu in liver and bone with decreased Zn and Fe in liver. Ivan and

Hidiroglou (1980) reported reduced average daily gain and higher feed

per unit gain when sheep were supplemented with 3000 ppm Mn from MnSO4.H20

compared with supplemental levels of 22 and 300 ppm. These workers also

found decreased liver Fe and Cu as dietary Mn increased.

Reid et al. (1947) reported negative Ca balance in lactating dairy

cows supplemented with 70 ppm Mn from MnSO4. Blakemore et al. (1937)

observed that spontaneous lactation tetany was seen in cows grazing

pastures high in Mn. When young cattle were fed manganese sulfate at

levels ranging from 250 to 2000 ppm Mn, fecal Ca and P were increased,

although positive Ca and P balances were found up to 500 ppm (Gallup

et al., 1952). Cunningham and coworkers (1966) added 0, 820, 2460, or

4920 ppm Mn to a basal diet for growing calves containing 12 ppm Mn.

The highest supplemental levels decreased growth and intake but did not

influence feed conversion, hemoglobin, or other serum constituents. A

similar experiment also using manganese sulfate and 0, 1000, 2000, or

3000 ppm for 100 days was conducted; however, feed intake was held

constant for all treatments (Cunningham et al., 1966). In this experi-

ment there was no effect on weight gain or feed conversion, but hemoglobin









was decreased by 3000 ppm Mn. These workers hypothesized that reduced

growth observed with high dietary Mn was due primarily to reduced feed

intake. They also conducted in vitro studies which indicated that high

dietary Mn changed the microbial population of the rumen and suggested

that the depressing effect on performance may be due in part to changes

in the rumen microflora. Similar changes on bacterial populations were

noted in the cecum of rats (Meghal and Nath, 1964) and in the ability of

rumen microorganisms from beef cattle to digest cellulose in vitro

(Robinson et al., 1960) when Mn was supplemented at high levels in these

animals. Robinson and coworkers (1960) supplemented MnS04 at levels up

to 1000 ppm in feedlot cattle. They found reduced Fe absorption and

fiber digestibility but no effect on Ca or P retention, hemoglobin, or

serum Ca, P, or Fe. They reproduced these results when Mn was fed at

levels up to 600 ppm (Robinson et al., 1961).

Manganese toxicosis in poultry was investigated by several groups.

Heller and Penquite (1937) observed reduced performance and 52% mortali-

ty in chicks fed 1% MnCO3 (approximately 4800 ppm Mn). Van der Hoorn

et al. (1938) reported increased mortality and reduced performance in

chicks fed .1% MnO2 (approximately 600 ppm) for 12 weeks. In contra-

distinction, Gallup and Norris (1939a) fed MnCO3 to chicks for 20 weeks

at levels up to 1000 ppm with no apparent adverse effect. Vohra and

Kratzer (1968) observed no toxicosis from MnSO4-H20 in turkey poults fed

for 21 days at levels up to 4080 ppm. When supplemental Mn reached

4800 ppm, however, there was growth depression but no mortality. Heller

and Penquite (1942) found reduced performance in chicks supplemented

with 4800 ppm Mn. More recently, Southern and Baker (1983a) fed four

reagent grade Mn sources to chicks at concentrations of 3000 to 5000 ppm.









They reported depressed weight gain from MnCl2.4H20 and depressed hemo-
globin from MnCl2.4H20, MnC03, and MnSO4.H20, but not MnO2.

Richards (1930) fed 3.5 g of manganese citrate to pigs daily for 9

months without any adverse effects. In contrast, Grummer et al. (1950)
reported that 500 ppm Mn as MnSO4.H20 fed to pigs retarded appetite and

growth, especially during the latter part of the trial. They also began
to exhibit stiffness of limbs and a stilted gait. In 1962, Leibholz and

associates fed manganese sulfate to young pigs in two trials. Data

indicated that neither total gain nor feed conversion was significantly

altered by dietary Mn intake up to 2025 ppm. There was evidence of re-

duced growth at the highest level fed (4000 ppm). Bunch et al. (1963,

1964) reported that 2000 ppm Mn as MnCO3 produced no differences in

weight gain or feed conversion in baby pigs.
Manganese poisoning in man (primarily in miners) merits mention in

respect to one aspect of metabolism not previously mentioned. The

similarity between the clinical features of the extrapyramidal disease

of chronic Mn intoxication (e.g., neurological and neurobehavioral dis-

orders) and Parkinson's disease led to the investigation of a relation-

ship between Mn and catecholamines. Papavasiliou et al. (1968) proposed

that cyclic AMP is the link between biogenic amines and Mn metabolism.
They showed that substances which altered cyclic AMP also altered Mn

metabolism. The features of manganism and parkinsonism both respond
favorably to administration of L-dopa (dihydroxyphenylalanine) (Mena

et al., 1970; Cotzias et al., 1971). Cotzias et al. (1972) extended

these studies to pallid mice which were found to differ from normal mice
in the metabolism of L-dopa and tryptophan as well as Mn. The exact

nature of this relationship requires further investigation.





-46-


Evidence indicates that dietary manganese is well tolerated by

livestock from several inorganic sources. The National Research Council

(1980) set maximum tolerable levels in animals on a well-balanced,

adequate diet. They indicated that cattle and sheep can tolerate 1000

ppm, at least under short term conditions. The poultry maximum is con-

siderably higher, at 2000 ppm, while some evidence suggests that swine

are less tolerant and the maximum dietary level was set at 400, although

higher levels are probably safe.














CHAPTER III

BIOLOGICAL AVAILABILITY OF REAGENT GRADE MANGANESE SOURCES AND
EFFECTS OF HIGH DIETARY MANGANESE IN BROILER-TYPE
CHICKS--EXPERIMENT 1


Introduction


An experiment was conducted with broiler-type chicks to study

tissue uptake of Mn as a measure of biological availability of Mn

sources and investigate possible toxic effects of high levels of die-

taryMn. A basal corn-soybean meal diet (116 ppm Mn) was supplemented

with 0, 1000, 2000, or 4000 ppm Mn as either reagent grade sulfate,

carbonate, or monoxide, and fed ad libitum for 26 d. Liver, heart,

skeletal muscle, and bone samples were excised and frozen for subse-

quent mineral analysis. No toxic effects were noted as expressed by

feed intake, body weight gain, feed conversion, hematocrit, hemoglobin,

or mortality. Analysis of Mn in tissues revealed a highly linear rela-

tionship between liver or bone Mn concentrations and dietary Mn for all

three sources. Manganese concentration in all tissues increased

(P < .01) as dietary Mn increased. Liver and bone Mn accumulation

appeared to be excellent indicators of relative biological availability.

Comparison of the three reagent grade sources revealed that on the

basis of tissue uptake and solubility tests, MnSO4.H20 was the most

available, followed by MnO and MnCO3, respectively.


-47-









Literature Review

Two diseases of poultry, perosis and nutritional chondrodystrophy,

are caused by a deficiency of Mn and may be prevented by Mn supple-

ments (Wilgus et al., 1937a;Lyons and Insko, 1937). Practical poultry

diets are now routinely supplemented with Mn. Relative biological

availability of Mn sources is therefore a practical and economic con-

cern when inherently low Mn feeds such as corn and sorghum comprise a

major portion of poultry diets (Underwood, 1981). Most of the biological

availability research with Mn sources has been conducted with poultry

(Ammerman and Miller, 1972). Early studies using reduction of the

incidence of perosis as the criterion (Schaible et al., 1938; Gallup

and Norris, 1939a) found most Mn sources except silicate and carbonate

ores equally available. Isotope studies (Hennig et al., 1967) revealed

greater availability in 54MnCl2 than 54MnSO4 or 54MnO2' More recently,

Watson et al. (1970, 1971) found that leg abnormality score was a more

sensitive, albeit somewhat subjective, criterion for availability of Mn

than body weight, bone ash, or bone Mn concentrations. These workers

suggested that a more sensitive assay might result if higher levels of

added Mn were supplied from test sources using bone Mn as the response

criterion.

It has been suggested (Miller, 1983) that biological availability

may best be determined by measuring some aspect of functional effective-
ness. It is necessary to feed a basal diet that is deficient in the

element in order to measure parameters of effectiveness such as growth

rate, remission or prevention of deficiency signs, and biochemical

changes. There are certain disadvantages to this technique such as









objectively quantifying effects (e.g., leg deformity score), the

increased cost of feeding purified diets, and possibility of errors
due to contamination. Using tissue Mn accumulation at high but non-

toxic levels as a measure of biological availability has the advan-

tages that natural diets may be fed which allow animals to reach full

genetic growth potential, any contamination would be relatively insig-

nificant, and modern atomic absorption techniques are sensitive and

accurately quantify tissue Mn concentrations.

Whenever mineral supplementation of animal feeds occurs, the

possibility of errors leading to toxicosis exists. Although Mn toxi-

cosis in several mammalian species has been documented (Underwood,

1977), there is little information regarding the toxic level of various

sources of Mn in modern poultry diets and no acute oral toxicity or

long-term feeding studies of MnO have been reported (Select Committee

on GRAS Substances, 1979). Manganese was tolerated at levels of 385 ppm

from MnSO4 and MnCO3 in poults (Mussehl and Ackerson, 1939) and 1000 ppm

as MnCO3 in chicks (Gallup and Norris, 1939a). However, Van der Hoorn

et al. (1938) reported increased mortality and reduced performance in

chicks fed .1% MnO2 (approximately 600 ppm) for 12 wk. Heller and

Penquite (1937) observed reduced performance and 52% mortality in

chicks fed 1% MnCO3 (approximately 4800 ppm Mn) while Vohra and Kratzer

(1968) found no toxic effects in turkey poults fed at a level of
4080 ppm. However, growth depression but no mortality resulted at a

level of 4800 ppm as MnSO4-H20. Southern and Baker (1983a) recently

fed four reagent grade sources to chicks at concentrations of 3000 to

5000 ppm. They reported depressed weight gain from MnCl2.4H20, and

depressed hemoglobin from MnCI2-4H20, MnC03, and MnSO4.H20, but not MnO2.









The present study was undertaken to investigate tissue uptake at

high dietary levels as an objective method for determining relative

biological availability of Mn from different sources in chicks and to

further elucidate toxic levels of dietary Mn.


Materials and Methods


One hundred twenty, day-old Cobb color-sexed male chicks were

randomly assigned to pens in a thermostatically controlled, electrically

heated Petersime battery with raised wire floors. Two replicates of

six chicks each were assigned to each treatment, and chicks were fed a

basal corn-soybean meal diet to 4 d posthatching. Chicks were main-

tained on a 24 h constant-light schedule and allowed ad libitum access

to feed and tap water. On d 4 posthatching, the chicks were weighed

and placed on the experimental diets for 26 d.

The basal diet (table 1) was a conventional corn-soybean meal diet
(116 ppm Mn) designed to meet or exceed the nutrient requirements of the

growing chick (National Research Council 1977). Manganese additions

were made to the diets at the expense of washed sand, to supplement at

0, 1000, 2000, or 4000 ppm Mn. The Mn sources (MnSO4.H20, MnCO3, and

MnO) were reagent grade chemicals. Chemical and physical character-

istics (table 2) of the sources were determined. Manganese concentra-

tion, and Ca, Mg, Zn, Fe, and Cu contaminants were determined by atomic

absorption spectrophotometry (Anonymous, 1982) on a Perkin-Elmer

Model 5000, and P by a modified colorimetric method (Harris and Popat,

1954). Relative solubilities (Watson et al., 1970) and magnetic

susceptibility (Watson et al., 1971) of the Mn sources were determined,

and X-ray diffraction patterns were interpreted.









At the termination of the experiment, final chick weights and

feed consumption were determined. Four chicks from each pen were ran-

domly selected for tissue analysis. Blood samples were taken by

anterior cardiac puncture and plasma saved for mineral analysis.

Hemoglobin was determined by a cyanmethemoglobin method (Unopette test

5857/5858; Becton-Dickinson, Rutherford, New Jersey), and hematocrit

by a micro-hematocrit method (Cohen, 1967). Chicks were sacrificed by

cervical dislocation and liver, pectoralis major muscle, heart, and

right tibia were excised for mineral analysis. Tissue Ca, Mg, Mn, Zn,

Fe, Cu, and P were measured in liver, muscle, and heart as indicated

previously. Zinc, Fe, and Cu were not measured in bone, nor was Fe in

plasma. Plasma Mn was determined by flameless atomic absorption

spectroscopy (Anonymous, 1974) using the Perkin-Elmer Model 503 with

HGA-2100 Graphite Furnace by the following method. A 1 to 5 dilution

of plasma in 10% (w/v) tricholoracetic acid (Fick et al., 1979) was

drawn into a 20 pl pipet which had previously been flushed with xylene

and injected into the graphite tube. Control settings were as follows:

gas flow, 35; slit, 4; deuterium lamp source; program functions (time/

temperature), dry 22 s/115 C; char 10 s/880 C; atomize 12 s/2700 C.

Standards were .005, .01, .02, .03, .04, and .05 ppm Mn and contained

20% serum matrix and 8% trichloroacetic acid (Fick et al., 1979).

All data were analyzed by analysis of variance (Steel and Torrie,

1980) and differences were separated by Duncan's (1955) multiple range

test.









Results

Analysis of mineral sources included in table 2 indicated Mn

concentrations of 32.58, 47.13, and 76.66% for the sulfate, carbonate,

and oxide sources, respectively. The reagent grade Mn sources were

relatively free of mineral contaminants. The sulfate was 100% soluble

in water and the carbonate and oxide were virtually insoluble. The

carbonate was only 30.3% as soluble in neutral ammonium citrate as the

sulfate and oxide sources and all forms were completely soluble in dilute

(.4%) HCI. The oxide was slightly insoluble in 2% citric acid. Solu-

bility did not appear to be related to particle size. Only the oxide

form had significant magnetic susceptibility and it did not appear to

be related to iron contamination. A high degree of purity was indicated

by X-ray diffraction.

Chick performance data (table 3) indicate there were no differ-

ences in average daily feed intake, average daily gain, or feed con-

version (overall mean values 84.4 g, 45.6 g, and 1.85, respectively)

due to treatment. Average daily feed intake was the lowest for the

4000 ppm sulfate treatment (81 g) but average daily gain was among the

highest (46 g) while feed/unit gain was among the lowest for this treat-

ment (1.76). Mortality was not significant, with only one death occur-

ring in one replicate of the 2000 ppm oxide treatment.

Hemoglobin and hematocrit were reduced numerically by all Mn sources
as dietary level increased (table 4), but the decreases were not sig-

nificant (P > .05). Plasma Mn increased (P < .01) from 3.74 pg/dl as
dietary level increased for all sources, although the 1000 ppm level

was not different from controls. The 4000 ppm level as sulfate (13.57









jg/dl) produced higher (P < .01) Mn levels in the plasma than did the

equivalent level from carbonate (9.1 pg/dl), with oxide (12.04 1jg/dl)

being intermediate (figure 2). Linear regression analysis (table 5)

indicated an inconsistent but linear increase in plasma Mn as dietary

levels increased. The sulfate produced the most linear response in

plasma as shown by the high correlation coefficient (r = .92). Plasma

Ca, P, and Mg averaged 11.5, 7.55, and 1.99 mg/dl and Zn and Cu 223 and

18.7 jig/dl and were not affected (P > .05) by dietary Mn (table 4).

Liver Mn increased (P < .01) as dietary Mn increased from all

sources (table 6, figure 3). Regression analysis (table 5) indicated

a highly linear uptake of liver Mn as dietary Mn increased with all

sources tested. Liver Mn concentrations at the 1000 ppm level from

sulfate (20.9 ppm) and oxide (17.8 ppm) were higher (P < .01) than the

unsupplemented control (11.6 ppm) and the 4000 ppm level from these

sources produced higher liver Mn (31.0 and 27.4 ppm) than did the same

level from the carbonate source (20.3 ppm). There was a numerical

reduction in liver Fe as sulfate supplementation increased, but this

trend was not evident in chicks fed the other sources. Liver Ca, P,

Mg, Zn, Fe, and Cu averaged 111, 10822, 648, 84.8, 256, and 14.9 ppm

and were not affected by dietary treatment.

Dietary Mn increased heart Mn concentration slightly (table 7)

but only the 2000 and 4000 ppm from sulfate (3.73 and 3.89 ppm) pro-

duced tissue levels that were higher (P < .01) than the control group

(2.15 ppm). Figure 4 indicates the inconsistent uptake of Mn in the

heart. Skeletal muscle (table 8) contained lower concentrations of Mn

than did cardiac muscle and only increased slightly (less than 2 ppm)

as dietary Mn increased. Only the 2000 and 4000 ppm levels from









sulfate and carbonate produced muscle Mn that was higher than controls

(P < .01). A more linear response was found for skeletal muscle (cf.

figure 5) than for cardiac muscle which is also evident in linear

regression analysis of the two tissues (table 5).

Bone ash was decreased by some treatments (P < .05) but no con-

sistent pattern was evident (table 9). Manganese in bone ash increased

(P < .01) in a highly linear nature as indicated by figure 6 and regres-

sion analysis (table 5) from all sources. The sulfate produced greater

Mn concentrations than the oxide or carbonate at both the 2000 and

4000 ppm level of supplementation (P < .01). The oxide produced higher

bone Mn than the carbonate form at the 4000 ppm level (P < .01). Bone

Mn was increased from 12.3 ppm in controls to 207.4, 78.6, and 129.8

ppm for the sulfate, carbonate, and oxide sources when supplemented at

4000 ppm. This amounts to greater than a 16x increase for the sulfate

source.



Discussion


Homeostatic mechanisms maintain tissue Mn levels at remarkably

constant levels over a wide range of dietary intakes (Underwood, 1977;

Miller, 1979). These mechanisms include both variable excretion

(Britton and Cotzias, 1966) and variable absorption (Lassiter et al.,

1974; Abrams et al., 1976). Results of the present study indicate

techniques available are able to differentiate biological availability

of Mn based on tissue uptake when sources are fed at high but nontoxic

levels in the chick. Tissue Mn concentration was increased (P < .01)

in all tissues studied when supplemented up to 4000 ppm. When dietary





-55-


Mn from MnSO4.H20 increased 35x, bone Mn increased 16x and liver in-

creased 2.7x. This is in contrast to rats in which liver Mn was
elevated by only .65x when the dietary level was increased 500x (Abrams

et al., 1976). Several factors influence Mn utilization (Wilgus and

Patton, 1939; Schaible and Bandemer, 1942; King et al., 1979; Rehnberg

et al., 1980) and these differences in liver uptake are not surprising,

given species and diet differences.

Liver and bone Mn concentrations increased linearly with increased

intake for all three Mn sources. Bone Mn (Watson et al., 1970;

Southern and Baker, 1983b) and bile (Southern and Baker, 1983a) had

previously been shown to increase linearly with increasing dietary

levels. The linear uptake of Mn in liver and bone make these tissues

useful measures of biological availability that are more objective than

leg deformity score (Watson et al., 1970, 1971). Differences in Mn

bioavailability are more readily apparent in tissue when Mn is fed at

high graded levels than with assays using purified diets supplemented

at levels below the requirement. This is indicated by higher correla-

tion coefficients for bone (r = .95, .88, and .90 for sulfate, carbonate,

and oxide, respectively) in the present study compared to r = .67 when
smaller increments (0, 10, 20, 30 ppm Mn) were added to purified diets

as MnSO4"H20 (Watson et al., 1970).

Skeletal and cardiac muscle were variable in Mn uptake (figures
4, 5) and contained relatively low Mn concentrations. Thus, muscle

uptake would not be a satisfactory measure of biological availability.

The higher concentration of Mn in cardiac muscle is a function of higher

mitochondrial concentration in cardiac as opposed to skeletal muscle,

since Mn tends to concentrate in mitochondria (Maynard and Cotzias, 1955).









Solubility of Mn in neutral ammonium citrate was highly correlated

to biological availability in chicks (Watson et al., 1971). By this

criterion the sulfate and oxide sources should be more available than

the carbonate, as confirmed by tissue uptake data. Based on bone and

liver uptake and solubility, MnSO4-H20 had the highest availability

followed by MnO and MnCO3 was the least available of the three reagent

grade sources tested.

Estimates of the relative value of the Mn sources may be determined

in several ways. Multiple linear regression of bone ash Mn concentra-

tion with respect to dietary levels yields the following equation:


y = 22.20 + .046362xI + .014649x2 + .027617x3 (r = .995),

where y equals bone Mn, ppm ash basis, x1 equals ppm Mn as sulfate,

x2 equals ppm Mn as carbonate, and x3 equals ppm Mn as oxide. Similarly,

multiple linear regression of liver Mn concentration with respect

to dietary Mn yields:


Y = 14.56 + .004352x1 + .001570x2 + .003339x3 (r = .968).

Estimates of relative biological availability (table 10) may be

obtained by a ratio of the slopes, which represent the rate of change

in tissue Mn with respect to change in dietary Mn. Setting MnSO4 H 20

at 100% gives 31.6% for MnCO3 and 59.6% for Mn0 using bone Mn concen-

trations or 36.1% and 76.7% using liver Mn concentrations. Alterna-

tively, slopes from individual regression lines (table 5) may be compared

in a similar manner. This gives relative biological availability esti-

mates of 33.1% and 57.4% for MnCO3 and MnO using bone or 45.2% and

84.0% for liver. If average increase in tissue Mn concentration over









basal is used for comparison, slightly higher estimates are obtained,

38.2% for MnCO3 and 65.6% for MnO in bone or 49.5% and 79.4% in liver.

All estimates yield the same rank order for the different sources. An
index may be calculated using the mean of the three individual estimates

to approximate relative value of the sources. The estimate index values

for bone (MnCO3--31.6%, 33.1%, 38.2%; MnO--59.6%, 57.4%, 65.6%) are,

thus, 34.3% for MnCO3 and 60.9% for MnO. The estimate index values for

liver are slightly higher (MnCO3--36.1%, 45.2%, 49.5%; Mn0--76.7%,

84.0%, 79.4%) yielding 43.6% for MnCO3 and 80.0% for MnO. In this

experiment bone produced the more conservative estimate indices.

Based on leg deformity score, Watson et al. (1971) found a reagent

grade MnSO4.H20 more available than rhodocrosite (a natural carbonate

ore) but not different from reagent grade MnCO3. The sulfate was also

more available than four oxide sources tested. X-ray diffraction pat-

terns revealed that the two more available oxides in their studies were

composed primarily of manganosite (MnO) while pyrulosite (Mn02) was

present in the less available oxides and predominant in the oxide of

lowest availability. Southern and Baker (1983a) found reagent grade

MnSO4.H20 more available than reagent grade MnCO3 or reagent grade MnO2

but similar to reagent grade MnCl2'4H20 based on bile Mn concentration.

The dioxide (MnO2) was less available than all other sources. When

they used reduction in hemoglobin as the criterion, MnCl2.4H20 and MnCO3

were more available than MnO2. Liver Mn concentration was higher with

MnSO4"H20 as the source than MnCl2-4H20 or MnO2. They fail to ade-

quately explain the apparent contradictions although MnO2 is consistently

less available than any other source. Availability estimates are

given as 102.1% for MnCl2-4H20, 100% for MnSO4.H20, 77% for MnCO3, and









28.9% for Mn02, using bile Mn slope ratios. The present study indi-

cated that reagent grade MnO (with no measurable Mn02) was more

available than MnCO3. This is consistent with earlier studies which

found availability of oxides related to relative proportions of MnO to

MnO2 (Watson et al., 1971).

King and coworkers (1979) did not detect differences in availability

between reagent grade MnCO3 or the much more soluble MnCl2 fed to rats

at low dietary levels (<3 ppm). Kayongo-Male et al. (1974a)reported

that MnSO4.H20, MnCO3, and MnO were equally available to weanling pigs

when 10 ppm Mn was added to a basal diet containing 16 ppm Mn.

Although there was no statistical evidence of Mn toxicosis in the

present study, several trends suggested marginal toxicosis at the

highest levels of supplementation. These included numerically reduced

hemoglobin and hematocrit as all sources of dietary Mn are increased; a

decreasing concentration of liver Fe, especially with the more available

sulfate source; and decreased dietary intake at the highest level of

sulfate supplementation. The present study using modern poultry diets

agrees with other studies indicating that Mn toxicosis from highly

available sources such as MnSO4"H20 will occur at levels between 4000

and 5000 ppm Mn when growth depression is the criterion (Vohra and

Kratzer, 1968; Southern and Baker, 1983a). Higher concentrations could

be tolerated with less available sources. Toxicosis could occur at

lower concentrations under other conditions such as increased Mn ab-

sorption (Southern and Baker, 1983b), extended feeding periods (Van der

Hoorn et al., 1938), or Fe deficiency (Pollack et al., 1965), and is

exacerbated with purified diets (Heller and Penquite, 1937).





-59-


This study indicated that Mn from reagent grade MnSO 4H 20, MnC03,

and MnO was well tolerated up to 4000 ppm by chicks fed nutritionally

balanced diets for periods up to 26 d. In addition, liver and bone Mn

uptake was a sensitive and objective method of determining relative

biological availability of Mn sources when fed at high but nontoxic

levels. Data indicated that MnSO4*H20 was more available than MnO, and

MnCO3 was the least available of the three sources tested. Assuming

toxicity from pure sources is correlated to absorption, i.e., relative

bioavailability, MnSO4.H20 is the most toxic, followed by MnO and MnCO3,

respectively.










TABLE 1. COMPOSITION OF BASAL DIET--EXP. 1, 2, AND 3

International
Item Feed No. %

Ingredient compositiona
Corn, ground 4-02-992 55.65
Soybean meal, dehulled 5-04-612 37.00
Poultry oil 4-00-409 2.50
Dicalcium phosphate 6-00-080 1.70
Limestone, ground 6-02-632 1.00
Microingredientsb .50
Salt, iodized .40
DL-methionine .25
Filler, washed sand 1.00

Total 100.00

Chemical composition c
Crude protein, % 23.0
Metabolizable energy, kcal/kg 3016.0
Ca, % .91
P, % .76
Mg, % d,e .18
Mn, ppm 105.7
Fe, ppm 580
Zn, ppm 58
Cu, ppm 12.5

aAs-fed basis.

blngredients supplied per kilogram of diet: vitamin A palmitate, 6600 IU;
vitamin D3, 2200 ICU; menadione dimethylpyrimidinol bisulfite, 2.2 mg;
riboflavin, 4.4 mg; pantothenic acid, 13.2 mg; niacin, 39.6 mg; choline
chloride, 499.4 mg; vitamin B12, 22 pg; ethoxyquin, .0125%; manganese,
60 mg; iron, 50 mg; copper, 6 mg; cobalt, .198 mg; zinc, 36 mg.
CDry matter basis. Crude protein and metabolizable energy calculated;
minerals determined by analysis.
dActual manganese (by analysis): exp. 1, 116 ppm; exp. 2, 89 ppm;
exp. 3, 112 ppm.
eManganese supplement added at the expense of equivalent weights of
washed sand.






TABLE 2 CHEMICAL AND PHYSICAL CHARACTERISTICS OF MANGANESE SOURCES--EXP. 1-6

Chemical constituents, dry matter basis Particle size, %
Manganese
source Mn Ca P Mg Zn Fe Cu +30a -30+ 100 -100

% -----------------------ppm-----------------------

Oxide, 76.66 21.2 --b 50.2 8.3 4.8 748 __b 38.1 61.9
reagent grade
Oxide, 58.21 135 750 1231 445 25,272 426 -- 12.8 87.2
feed grade
Sulfate, 32.58 44.7 -- 51.8 22.7 118 -- 1.2 52.2 46.6
reagent grade
Carbonate, 47.13 47.0 -- 53.0 15.7 186 .. .. 4.8 95.2
reagent grade

Relative solubility, %c

Neutral Magnetic Interpretation
Physical ammonium HCl Citric susceptibility, of X-rayd
appearance Water citrate 0.4% acid, 2% % patterns

Oxide, Dark green, .4 100.0 100.0 92.0 84.6 MnO (reagent
reagent grade fine granules grade)
Oxide, Dark brown, 1.0 78.0 80.9 100.0 99.8 MnO plus minor
feed grade fine powder Mn30
Sulfate, Light pink, 100.0 100.0 100.0 100.0 2.3 MnSO4H2O
reagent grade crystalline powder (reagent grade)
Carbonate, Light tan, .8 30.3 100.0 100.0 1.8 MnCO3 (reagent
reagent grade fine powder grade)


aRetained by a No. 30 sieve (U.S. Bureau of Standards).

bNone detected.


CRelative to solubility in concentrated HCl at
37C for 1 hour, constant stirring.
dCourtesy of Agricultural Operations, Minerals Division,
International Minerals & Chemical Corporations,
Bartow, Florida.








TABLE 3. EFFECTS


OF SOURCE AND LEVEL OF DIETARY MANGANESE ON
AND FEED CONVERSION IN CHICKS--EXP. a


FEED INTAKE, GAIN,


Treatment
Added Mn Avg. initial Avg. daily Avg. daily Feed/unit
Mn, ppmb source weight, gC intake, gd gain, g gain


Interaction effects
0 -- 84.4 85.3 45.8 1.86
1000 sulfate 87.7 84.0 44.5 1.89
2000 sulfate 87.7 85.5 46.4 1.84
4000 sulfate 87.0 81.0 46.0 1.76
1000 carbonate 90.8 83.8 44.2 1.90
2000 carbonate 86.1 86.1 44.8 1.92
4000 carbonate 78.3 84.9 46.5 1.82
1000 oxide 80.7 85.1 45.4 1.87
2000 oxide 88.3 81.5 46.5 1.75
4000 oxide 89.6 85.4 45.7 1.87

SE 1.24 .55 .26 .02

a Each value represents the mean of 8 chicks per pen, fed diets for 26 days.

bBasal diet contained 116 ppm Mn.

cChicks placed on experimental diets at four days of age.

dAs-fed basis.







TABLE 4. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON HEMOGLOBIN, HEMATOCRIT, AND
MINERAL COMPOSITION OF PLASMA IN CHICKS--EXP. a

Mineral concentration
Treatment
Hemoglobin Hematocrit mg/dl pg/dl
Addedb Mn
Mn, ppm source g/dl % Calcium Phosphorus Magnesium Manganese Zinc Copper

Interaction effects

0 -- 8.50.38 29.9 .59 11.4.25 7.58.18 1.93.07 3.74 .22 c 20712 17.6 .8
1000 sulfate 8.05.21 28.4 .52 11.3.47 7.62.15 2.12.12 6.34 .29c'd 202 17.61.3
2000 sulfate 7.90.41 27.5 .48 11.5.39 7.67.18 1.98.04 8.41+ .99d'e 21614 18.31.2
4000 sulfate 7.43.28 27.8 .52 11.1.53 7.23.14 1.92.08 13.57 .72g 228 9 19.0 .9
1000 carbonate 7.75.24 28.8 .64 12.1.42 7.56.17 2.23.13 6.45 cd 24411 20.5+1.7
2000 carbonate 7.50.33 28.4 .67 11.3.49 7.53.19 1.94.08 7.57 42'e f 22411 19.11.6
4000 carbonate 7.38.42 27.4 .37 11.4.39 7.64.17 1.98+.08 9.10 .71_' 23511 18.01.3
1000 oxide 7.78.27 29.01.00 11.1.26 7.46.13 1.96.06 6.25+ .12c d 229 9 17.41.5
2000 oxide 7.53.50 27.7 .35 11.7.37 7.59.14 1.97.09 10.87+1.03 e f g 10 19.91.0
4000 oxide 7.63.31 27.81.34 11.8.47 7.57.13 1.87.06 12.04 .22Tfg 22611 19.51.5

aEach value represents the mean SE of 8 chicks per treatment, fed diets for 26 days.

bBasal diet contained 116 ppm Mn.

c'd'e'f'gMeans in the same column with different superscripts are significantly (P < .01) different.











TABLE 5. LINEAR REGRESSION ANALYSIS OF TISSUE MANGANESE WITH
RESPECT TO DIETARY MANGANESE--EXP. 1
Mn Correlation
source Regression equation coefficient

Livera


y = 13.93 + .00456x
y = 13.11 + .00206x
y = 13.09 + .00383x

Hearta

y = 2.61 + .000386x
y = 2.57 + .000071x
y = 2.32 + .000196x

Musclea

y = .81 + .000376x
y = .75 + .000403x
y = .33 + .000168x

Bonea

y = 15.41 + .0486x
y = 17.88 + .0161x
y = 21.47 + .0279x

Plasmab

y = 3.75 + .00244x
y = 4.52 + .00126x
y = 4.50 + .00212x


aWhere y equals tissue
ppm. Each regression


manganese, ppm, and x equals dietary manganese,
equation represents 32 chicks.


bWhere y equals plasma manganese, Vg/dl, and x equals dietary manganese,
ppm. Each regression equation represents 16 chicks.


Sulfate
Carbonate
Oxide



Sulfate
Carbonate
Oxide


Sulfate
Carbonate
Oxide



Sulfate
Carbonate
Oxide


Sulfate
Carbonate
Oxide







TABLE 6. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE MINERAL
COMPOSITION OF LIVER IN CHICKS--EXP. Ia

Treatment Mineral concentration (ppm, dry matter basis)

Added b Mn
Mn, ppmb source Calcium Phosphorus Magnesium Manganese Zinc Iron Copper

Interaction effects
0 -- 90 8 10,100543 64415 11.6+ .61 80.04.0 27632 15.1 .6
1000 sulfate 13016 10,923177 658 5 20.9 .59f,e,f 89.43.0 29628 15.81.0
2000 sulfate 115 7 11,348105 67714 24.21.13 87.92.2 22710 15.8 .8
4000 sulfate 11212 11,195183 643 7 31.02.31 d 80.62.3 205 6 14.0 .8
1000 carbonate 11014 10,667288 61621 16.1+ 53'd 83.81.2 27253 13.9 .5
2000 carbonate 12418 11,033172 64914 18.9 .70 ,eef 86.6+2.2 32725 16.2 .8
4000 carbonate 11215 10,576564 65013 20.3 .88d ,e 81.1.2 24621 14.71.0
1000 oxide 11012 10,578359 64112 17.8+ .71 ,f 82.31.6 23431 14.81.1
2000 oxide 11213 10,891293 66913 22.4 'h 89.83.9 24620 14.8 .5
4000 oxide 99 6 10,908229 63616 27.42.11gh 85.72.6 23021 14.1 .4

aEach value represents the mean + SE of 8 chicks per treatment, fed diets for 26 days.

bBasal diet contained 116 ppm Mn.

cdefghMeans in the same column with different superscripts are significantly (P < .01) different.







TABLE 7. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE
MINERAL COMPOSITION OF HEART IN CHICKS--EXP. a

Treatment Mineral concentration (ppm, dry matter basis)

Added b Mn
Mn, ppmb source Calcium Phosphorus Magnesium Manganese Zinc Iron Copper

Interaction effects
0 -- 206t 9 7,689296 83223 2.15.22 95.03.1 23230 18.7 .7
1000 sulfate 21826 6,768301 80736 3.39.28d 92.55.3 22517 19.31.2
2000 sulfate 183 5 6,746306 80224 3.73.36 ,e 93.03.9 23313 18.41.3
4000 sulfate 181 5 6,961169 81117 3.89+.23,d 96.12.0 18819 18.31.2
1000 carbonate 193 3 6,705217 79628 3.06.28c de 103.44.9 25022 21.6 .9
2000 carbonate 185 7 7,022242 81024 2.93.43, d,e
4000 carbonate 191 7 6,783191 84217 2.61.29,d e 104.3+2.8 20510 20.51.1
1000 oxide 182 7 6,520377 79832 2.93-.31d 100.54.9 19210 20.41.4
2000 oxide 186 8 6,113184 81320 2.40+.31,d 1 93.53.3 21310 20.91.4
4000 oxide 176 4 6,524213 770t32 3.14.34 94.34.1 26053 18.81.4

aEach value represents the mean SE of 8 chicks per treatment, fed diets for 26 days.

bBasal diet contained 116 ppm Mn.

cdeMeans in the same column with different superscripts are significantly (P < .01) different.







TABLE 8. EFFECTS OF SOURCE AND LEVEL OF DIETARY MANGANESE ON THE MINERAL
COMPOSITION OF MUSCLE IN CHICKS--EXP. a

Treatment Mineral concentration (ppm, dry matter basis)

Added b Mn
Mn, ppm source Calcium Phosphorus Magnesium Manganese Zinc Iron Copper

Interaction effects

0 -- 79.4 8 8,273 351 930 40 .79.04 c 19.11.1 13.71.0 2.75.4
1000 sulfate 78.5 4 8,080 166 989 36 1.10.06 e 20.41.3 14.6 .6 2.70.3
2000 sulfate 70.6 5 7,874 184 923 20 1.71+.20 ,e 19.1 .9 13.8 .9 2.57.2
4000 sulfate 64.9 4 8,070 291 910 25 2.25+.2,e 18.8 .7 13.01.3 2.84.5
1000 carbonate 86.4 6 10,1841,532 1,027 33 1.02+.07 ,d 18.7 .5 15.61.7 4.02.8
2000 carbonate 76.9 6 9,5481,193 1,105103 1.72.26 e 19.2 .4 13.8 .8 2.93.2
4000 carbonate 90.015 7,903 588 948 21 2.31+.35e 19.1 .6 14.0 .9 2.92.4
1000 oxide 80.2 7 8,720 819 1,003 43 1.21.16c d 19.4 .6 14.0 .9 3.50.6
2000 oxide 77.8 8 9,7361,211 1,029 23 1.40.13 18.8 .4 17.82.7 3.79.6
4000 oxide 67.4 4 8,184 578 920 28 1.52.17 ,e 16.8 .6 12.1 .9 1.85.2

aEach value represents the mean SE of 8 chicks per treatment, fed diets for 16 days.

bBasal diet contained 116 ppm Mn.

cdeMeans in the same column with different superscripts are significantly (P < .01) different.







TABLE 9. EFFECT OF DIETARY MANGANESE ON BONE ASH AND MINERAL
COMPOSITION OF BONE IN CHICKS--EXP. Ia

Mineral concentration
Treatment Bone
ash, % %, ash weight basis ppm, ash weight basis
Added b Mn____________
Mn, ppmb source (dry, fat-free bone) Calcium Phosphorus Magnesium Manganese

Interaction effects
0 -- 46.9.77c 37.8.43 17.3.35 .706.034 12.3 "82f h
1000 sulfate 43.1.86e 37.3.53 17.7.18 .734.023 65.1+ 7.05g'
2000 sulfate 45.4.69c d'e 36.4.57 17.7.17 .722.021 117.210.52.
4000 sulfate 43.9.76 de 36.8.49 17.5.22 .735.030 207.4+13.55J
1000 carbonate 44.6.87c d'e 37.6.54 17.4.23 .706.027 37.8+ 4 f52g'1
2000 carbonate 45.4.64cde 37.1.41 17.3.27 .731.026 55.5+ 4.90g'
4000 carbonate 45.8.77cd 37.0.64 17.4.21 .695.029 78.6 5.80
1000 oxide 44.5.58'a'e 36.7.65 17.1.37 .729.022 61.4, 6.95g'n
2000 oxide 43.5.29de 37.0.59 17.4.21 .719.028 77.4+ 6.45h
4000 oxide 45.2.76c'de 36.9.40 17.4.36 .711.019 129.8 9.551

a Each value represents the mean SE of 8 chicks per treatment, fed diets for 26 days.

bBasal diet contained 116 ppm Mn.

cdeMeans in the same column with different superscripts are significantly (P < .05) different.

f'g'h'i'JMeans in the same column with different superscripts are significantly (P < .01) different.









TABLE 10. RELATIVE BIOLOGICAL AVAILABILITY OF THREE MANGANESE SOURCES BASED ON
MULTIPLE LINEAR REGRESSION, LINEAR REGRESSION, AND TISSUE MANGANESE CONCENTRATION--EXP. 1

Multiple linear Relative Linear Relative Avg. tissue Relative
Source regression slope value, % regression slope value, % Mn increase value, %

Bone

Sulfate .0464 100.0 .0486 100.0 117.6a 100.0
Carbonate .0146 31.6 .0161 33.1 44.9 38.2
Oxide .0276 59.6 .0279 57.4 77.2 65.6

Liver

Sulfate .00435 100.0 .00456 100.0 13.76b 100.0
Carbonate .00157 36.1 .00206 45.2 6.81 49.5
Oxide .00334 76.7 .00383 84.0 10.92 79.4

aparts per million, ash weight basis.

bparts per million, dry matter basis.










15

0
10
~C03



0,.



0 1000 2000 4000
ADDED DIETARY Mn, PPM



FIGURE 2. Effect of level and source of dietary Mn on plasma Mn concentration in
chicks--Exp. 1










30 04-'4
m 0


O 20 C03



-10
-J




0 1000 2000 4000
ADDED DIETARY Mn, PPM



FIGURE 3. Effect of level and source of dietary Mn on liver Mn concentration in
chicks--Exp. 1









4 S04


03 0
co3
0.


I-






0 1000 2000 4000
ADDED DIETARY Mn, PPM



FIGURE 4. Effect of level and source of dietary Mn on heart Mn concentration in
chicks--Exp. 1









2.5
C03

02.0

-1.5 0


w
.11.0


2 1.5

I I I
0 1000 2000 4000
ADDED DIETARY Mn, PPM



FIGURE 5. Effect of level and source of dietary Mn on muscle Mn concentration in
chicks--Exp. 1























0 1000 2000
ADDED DIETARY Mn, PPM


FIGURE 6. Effect of level
chicks--Exp. 1


and source of dietary Mn on bone Mn concentration in


200

m
4150


100

2
z
0


4000














CHAPTER IV

EFFECTS OF HIGH, GRADED DIETARY LEVELS OF REAGENT GRADE
MANGANOUS MONOXIDE ON TISSUE MINERAL COMPOSITION OF
BROILER-TYPE CHICKS--EXPERIMENT 2


Introduction

The relationship between high, but nontoxic, dietary levels of

manganous monoxide (MnO) and tissue mineral composition was investigated

in an experiment conducted with broiler-type chicks. A basal corn-

soybean meal diet (89 ppm Mn) was supplemented with 0, 1000, 2000, or

3000 ppm Mn as reagent grade MnO and fed ad libitum for 22 d. There

were six replications per treatment and six chicks per replication.

Liver, kidney, skeletal muscle, and bone were excised from four chicks

per replication. Plasma from the remaining two chicks was pooled and

all samples were frozen for subsequent mineral analysis. There were no

differences in average daily feed intake, average daily gain, or feed

conversion among treatments. Liver, kidney, bone, plasma (P < .01),

and muscle (P < .05) Mn concentrations increased as dietary Mn increased.

The relationship between dietary Mn and tissue Mn was linear (P < .01)

for all tissues. Supplementation at 2000 and 3000 ppm Mn reduced Fe

in the liver (P < .05) relative to the 1000 ppm dietary level. Data

indicated that bone and kidney provided more sensitivity for bioassay

than liver, muscle, or plasma. Reagent grade MnO was nontoxic when

fed at levels 55x the minimum requirement.


-75-









Literature Review


The average concentration of Mn in the animal body is approximately

.25 ppm (Underwood, 1977; Miller, 1979), and this small quantity is

widely, though unevenly, distributed throughout tissues and fluids.

Manganese concentration in tissues is relatively constant among species,

and in individual animals under normal dietary conditions (Doyle and

Spaulding, 1978), with concentrations varying between .5 and 12 ppm.

Manganese is more concentrated in mitochondria than other cell organelles

and therefore tends to be higher in tissues rich in mitochondria (Maynard

and Cotzias, 1955; Theirs and Vallee, 1957). Bone, liver, kidney, pan-

creas, and pituitary have high Mn concentrations while muscle tissue

contains very little.

Efficient homeostasis maintains tissue Mn levels at relatively con-

stant concentrations under varying dietary conditions but can be raised

or lowered by substantially increasing or decreasing Mn intakes

(Hidiroglou, 1979a; Underwood, 1981). Manganese concentration in bone

(Watson et al., 1970, 1971; Southern and Baker, 1983b; Exp. 1), bile

(Southern and Baker, 1983a), liver, and plasma (Exp. 1) of chicks was

increased linearly by increasing dietary Mn. In each of these experi-

ments with chicks, there were only two or three replications of each

treatment.

The Select Committee on GRAS Substances of the Life Sciences

Research Office (1979) reported in an extensive review that while MnO

supplies 90% of the Mn in animal feeds, there is insufficient data upon

which to evaluate it as a GRAS feed ingredient. No acute oral toxicity

studies or long-term feeding studies of MnO have been reported.









The present study was designed with six replications of each

treatment, to provide a sound statistical base for further investi-

gating the relationship between dietary Mn and Mn uptake of bone, liver,

kidney, skeletal muscle, and plasma in chicks fed high, graded levels

of reagent grade MnO; to determine the relative value of various tissues

for bioassay; and to evaluate oral toxicity of MnO at levels more than

55x the minimum requirement.


Materials and Methods


One hundred forty-four, day-old Cobb feather-sexed male chicks

were randomly assigned to 24 pens in a thermostatically controlled,

electrically heated Petersime battery with raised wire floors. Six

replicates of six chicks were assigned to each treatment, weighed at

d 0, and placed on experimental diets. The chicks were maintained on

a 24 h constant-light schedule, and allowed ad libitum access to feed

and tap water.

The basal diet (table 1) was a conventional corn-soybean meal diet

(89 ppm Mn) designed to meet or exceed nutrient requirements of the

growing chick (National Research Council, 1977). Reagent grade man-

ganous monoxide was added to the basal diet at the expense of washed

sand to provide 0, 1000, 2000, or 3000 ppm Mn. The chemical and

physical characteristics were determined previously (Exp. 1) and are

shown in table 2.

On d 22 posthatching, chicks were individually weighed and feed

consumption for each replication was determined. Four chicks from each

pen were randomly selected for tissue analysis. Blood was obtained from









the remaining two chicks by anterior cardiac puncture, pooled, and

plasma saved for mineral analysis. Chicks were sacrificed by cervical

dislocation and liver, kidney, pectoralis major muscle, and right tibia

were excised and frozen in heat-sealed polyethylene bags for subsequent

mineral analysis. Tissue Ca, Mg, Mn, Zn, Fe, Cu, and P were measured

as indicated previously (Exp. 1).

All data were analyzed by analysis of variance and regression

analysis by the least squares method with a single degree of freedom

regression model (Steel and Torrie, 1980). Differences between treat-

ment means were determined by Duncan's (1955) multiple range test.


Results

Chick performance data (table 11) indicated no differences in

average daily feed intake, average daily gain, or feed conversion due

to treatment (37.7 g, 24.6 g, and 1.57, respectively). A nonsignifi-

cant decreasing trend was observed, however, for intake, gain, and feed

conversion as dietary Mn increased from 1000 to 3000 ppm Mn.

Tissue mineral analysis (table 12) indicated that liver Mn con-

centration increased (P < .01) from 11.3 ppm (dry matter basis) in the

unsupplemented control to 18.7 ppm at the 3000 ppm dietary level.

Linear regression analysis (table 13) indicated that this increase was

linear (P < .01) with a correlation coefficient, r = .832. This is
illustrated in figure 7 which shows the plot of the prediction equa-

tion (y = 12.378 + .00235x) and the actual treatment means standard

errors. Liver Fe was reduced (P < .05) from 336 ppm at the 1000 ppm Mn
level to 242 ppm at the 3000 ppm level. Liver Ca, P, Mg, Zn, and Cu

concentrations were not influenced by dietary Mn.









Kidney Mn (table 12) was increased (P < .01) from 8.9 ppm Mn in
the control group to 28.7 ppm at the 3000 ppm level. In the control

group, kidney Mn was numerically lower than liver Mn but kidney Mn

increased at 2.75x the rate of liver Mn as dietary Mn increased.

Linear regression analysis (table 13) yielded the equation (y = 10.489

+ .00647x, r = .933) plotted in figure 8 and indicated the linear

(P < .01) increase in kidney Mn as dietary Mn increased. Neither Fe

nor other minerals in the kidney were affected by dietary Mn.

Manganese in muscle (table 12) was increased (P < .05) by dietary

Mn from .91 ppm at the 0 supplemental level to 1.38 ppm at the 3000 ppm

level. This increase was determined to be linear (P < .01) by regres-

sion analysis (table 13, figure 9); however, the low correlation coef-

ficient (r = .642) indicated only about 41% of the variance in muscle

Mn was due to regression on dietary level. The rate of increase in

kidney Mn was 40x that of muscle. No other muscle mineral concentra-

tions were affected by dietary Mn.

Bone ash (table 14) was not affected by dietary Mn and averaged

42.9% of the dry, fat-free bone. Manganese in bone ash (table 14)

increased linearly (P < .01) from 9.5 ppm to 67.4 ppm as supplemental

Mn increased from 0 to 3000 ppm. The regression equation (y = 8.657 +

.01935x) plotted in figure 10 had a correlation coefficient of r = .974

indicating the close relationship between dietary Mn level and bone Mn

concentration. The rate of uptake in bone was nearly 3x greater than

kidney, and 120x greater than muscle. Calcium, P, or Mg concentrations

were not affected by dietary Mn.

Plasma Mn (table 14) concentration increased (P < .01) from 3.37

pg/dl to 10.04 pg/dl as dietary Mn increased. This increase was linear








(P < .01) and is shown with the regression equation, y = 3.578 +

.00225x (figure 11). The correlation coefficient was r = .893 which

was lower than bone or kidney, but higher than liver or muscle. Plasma

Ca, P, Mg, Zn, or Cu concentrations were not affected by dietary Mn.


Discussion

The use of tissue Mn accumulation for a bioassay in chicks has

been investigated (Watson et al., 1970, 1971; Exp. 1). An ideal bio-

assay criterion should be linear or transformed to linear where

applicable, have a relatively steep slope, and a relatively low

standard deviation (S.M. Free, personal communication). These pro-

perties allow the use of slope of regression equations divided by

standard deviation (X criterion) to provide an index to the relative

power of various criteria for use in bioassay, when a linear relation-

ship exists between the independent and response variables. The purpose

of this experiment was to investigate the relationship between tissue

Mn accumulation and high dietary supplementation of reagent grade MnO,

determine the relative power of various tissues for bioassay, and

evaluate oral toxicity of MnO at levels more than 55x the minimum

requirement.

Manganese concentration in all tissues in the present study

exhibited a linear relationship (P < .01) with respect to supple-

mentary dietary levels. This is in agreement with other studies citing

evidence of a linear response in bone (Watson et al., 1970, 1971; Exp.

1; Southern and Baker, 1983b), liver, plasma (Exp. 1), and bile

(Southern and Baker, 1983a).








The linear uptake of tissue Mn with increased dietary Mn allows

calculation of A values for tissue accumulation (table 15). Based on

x values, the tissue of choice for bioassay would be bone followed by

kidney, plasma, liver, and muscle. The same ranking is obtained using

correlation coefficients (table 15) for comparison. In view of the

relative ease with which soft tissue Mn may be determined compared to

Mn in bone ash (appendix table A.1), it has been proposed (Exp. 1)

that soft tissue Mn accumulation (e.g., liver) can be a sensitive

method for determining relative bioavailability. Results from the

present study indicated that kidney Mn accumulation was a more sen-

sitive bioassay criterion than liver, muscle, or plasma. Plasma was

more sensitive than liver or muscle, although inconsistent correla-

tions were observed in Exp. 1.

Unsupplemented controls had a higher Mn concentration in liver

than kidney (11.3 vs 8.9 ppm). Since kidney accumulated Mn at a higher

rate than liver, supplementation raised the Mn concentration of kidney

above that of liver (28.7 vs 18.7 ppm at 3000 ppm dietary level). This

observation may explain why most reports (see Doyle and Spaulding,

1978) list liver Mn as more concentrated than kidney although Kato

(1963) and Rojas et al. (1966) using mice and rats, respectively, found

that the kidney was more greatly enriched with radioactive Mn than

liver. These studies and data from the present study indicate that
liver Mn concentration is more dynamic and transitory while kidney may

have a longer turnover time and/or greater storage capacity, as sug-

gested by Papavasiliou and coworkers (1966).

There was no evidence of Mn toxicosis other than a nonsignificant

trend suggesting decreased average daily intake and gain, and lower









feed efficiency. There was a depression in liver Fe as dietary Mn
increased from 1000 to 3000 ppm. This supports the hypothesis that

Mn toxicosis in chicks occurs at levels greater than 4000 ppm Mn from

highly available sources (Vohra and Kratzer, 1968; Southern and Baker,

1983a; exp. 1), which is more than 70x the minimum dietary level of

55 ppm (National Research Council, 1977). The reduction of liver Fe by

dietary Mn confirms earlier reports with several species (Hartman et al.,

1955; Theirs and Vallee, 1957; Robinson et al., 1960; Standish et al.,

1971) of the antagonistic effects of dietary Mn and Fe. No other
mineral interactions were observed in this experiment.

Variable absorption and excretion are involved in tissue Mn

homeostasis (Miller, 1973; Abrams et al., 1976). High dietary levels

of Mn can overload the enterohepatic circulation and exceed excretion

via bile (Cotzias, 1958), duodenal and jejunal mucosa (Papavasiliou et

al., 1966; Bertinchamps et al., 1966), and pancreas (Burnett et al.,

1952). Hall and coworkers (1981) cite evidence that the capacity of

liver to deal with large quantities of Mn is short-lived and when this

capacity is exceeded, plasma Mn concentrations increase. This leads to

accumulation in tissues. The present study suggests that dietary levels

for bioassay should be high enough to produce accumulation of Mn but not

high enough to induce toxicosis.
The minimum dietary Mn level for bioassay may be approximated.

Since kidney Mn is normally slightly lower than liver Mn, and greater

than liver Mn when the diet is supplemented with high levels, tissue Mn

accumulation has begun when kidney and liver have equal Mn concentrations.

Equating the regression equations for liver and kidney (12.378 +

.00235x = 10.489 + .00647x) and solving for x yields 458 ppm. Thus








the minimum dietary level for bioassay should be approximately 500 ppm
Mn. This recommendation is supported by the observation that all soft

tissues in unsupplemented controls in the present experiment had

analyzed Mn concentrations below that predicted by regression. Vari-

able absorption up to the point of saturation (Hall et al., 1981) would

yield curvilinear responses at low dietary levels. Setting the minimum

level at 500 ppm will remove this curvilinear effect and increase the

goodness of fit of the linear models and corresponding correlation coef-

ficients. Since the standard deviation (SD) tended to increase at

higher dietary levels in this study and Exp. 1, maximum bioassay levels

should be less than 3000 ppm, even though toxicosis was not observed

at 3000 ppm dietary Mn. A reduction in SD will increase X values and

relative bioassay power. Thus, a tissue accumulation bioassay in chicks

should be conducted with dietary Mn between 500 and 2500 (or less) ppm.

Data from the present study indicate that tissue uptake in response

to dietary level is linear and provides a useful, objective bioassay.

Bone Mn concentration would be the criterion of choice; however, kidney

Mn concentration is a sensitive measure and may be used for a more

rapid and less expensive assay procedure. In addition, dietary Mn

as reagent grade MnO is relatively non-toxic and is well-tolerated by

chicks at levels more than 55x the minimum requirement for 22 d.









TABLE 11. EFFECTS OF DIETARY MANGANESE ON FEED INTAKE, GAIN, AND
FEED CONVERSION IN CHICKS--EXP. 2a

Treatment
Avg. initial Avg. daily Avg. daily Feed/Unit

Added Mn, ppmb weight, gC intake, gd gain, g gain

0 48.4.61 38.31.20 25.0.93 1.56t.022

1000 48.9.67 38.81.35 25.1.87 1.55.020

2000 48.6.80 37.61.30 24.8.72 1.56.022

3000 48.3.57 36.2 .73 23.3.43 1.60.022

aEach value represents the mean SE of 6 pens, 6 chicks per pen, fed diets for 22 days.

bBasal diet contained 89 ppm Mn. Supplemental Mn as reagent grade MnO.

cChicks placed on experimental diets on day of birth.

dAs-fed basis.







TABLE 12. EFFECT OF DIETARY MANGANESE ON THE MINERAL COMPOSITION OF
LIVER, KIDNEY, AND MUSCLE IN CHICKS--EXP. 2a

Treatment Mineral concentration (ppm, dry matter basis)

Added Mn, ppmb Calcium Phosphorus Magnesium Manganese Zinc Iron Copper

Liver
0 39629 9,969521 71621 11.3 .53 79.33.7 29223fg 16.6 .6
1000 48144 9,689364 75226 16.1 .41d, 79.44.1 33625 19.01.0
2000 44438 9,807495 69424 17.5 .80 ,e 76.22.6 25217f 17.01.4
3000 43437 9,647432 66733 18.7 .80e 75.03.3 24213 16.61.2

Kidney
0 72446 9,212568 77513 8.9 .38' 69.81.9 207 9 16.6 .8
1000 71225 9,659425 75021 18.81.09e 74.22.9 20816 17.7 .8
2000 76033 9,625203 78318 24.41.03e 79.13.1 19210 17.11.0
3000 73849 9,480438 73915 28.71.49e 72.8 .5 231 6 17.2 .8

Muscle
0 25221 8,466325 1,15426 .91.05f 19.9 .7 34.42.9 5.4 .3
1000 255 8 8,451228 1,08639 1.12.05 ,g 22.0 .4 37.62.2 5.0 .4
2000 24013 8,252249 1,04940 1.33.15g 20.7 .7 34.72.1 5.7 .4
3000 236 8 8,229 62 1,05426 1.38.09g 20.7 .5 31.82.8 5.8 .3

a Each value represents the mean SE of 6 pens, 4 chicks per pen, fed diets for 22 days.

bBasal diet contained 89 ppm Mn. Supplemental Mn as reagent grade MnO.

cdeMeans in same column for a given tissue with different superscripts are significantly (P < .01) different.

f'gMeans in same column for a given tissue with different supercripts are significantly (P < .05) different.