Title: Manganese utilization by ruminants and poultry
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Title: Manganese utilization by ruminants and poultry
Physical Description: 109 leaves : ill. ; 28 cm.
Language: English
Creator: Watson, Larry Thomas, 1945-
Copyright Date: 1970
 Subjects
Subject: Feeds -- Composition   ( lcsh )
Manganese -- Physiological effect   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Larry Thomas Watson.
Thesis: Thesis (Ph. D.)--University of Florida, 1970.
Bibliography: Includes bibliographical references (leaves 102-108).
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098403
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000402702
oclc - 37711217
notis - ACE8626

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MANGANESE UTILIZATION BY RUMINANTS

AND POULTRY













By
LARRY THOMAS WATSON













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















UNIVERSITY OF FLORIDA
1970















ACKNOiT.EDCGELM1NTS


Sincere appreciation is extended to Dr. C. B. Arnmerman, Chairman

of the Supervisory Committee, for his thoughtful guidance, patience,

and constructive criticism which he gave to the author throughout the

academic program, experimental investigation and preparation of this

dissertation. The author also gratefully acknowledges the time and

as int. ce provided by the otlhe monmbors of the -uperviscry corrittee

including lrs. J. E. Moore, G. E. Combs, Jr., P. H. Hamnm, J. P. Feaster

and C. M. Allen, Jr.

The author is grateful for the assistance given him by.r;~,y of the

graduate students, particularly uillian G. lillis, Karl Fick, Jl 'a: P.

Staudish and Gladys Verde, and faculty rmembrs cf the rD.partrment of

Animal Science. Sincere appreciation is also expressed for the technical

assistance of Mrs. Sarah Miller and other mnoibers of the staff of tI-,

Nutrition Laboratory.

Special appreciation is expressed to South.eac!.ern Minerals Inc.,

Bainbridge, Georgia, Int'rn;ationala iineials and Chemical Corporation,

Skokie, Illinois,and the Center for Tropical Agricultrre, University

of Florida for partial financial support of this investigation. The

provision of expe:rincntal supplies by Monsanto Chemical Conpany,

St. Louis, Missouri ,and Dar.'e's Laboretories, Inc. Chicago, Tllinois is

gratefully acl'knoxlcddgd.








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

assistance in laboratory analyses and for typing this dissertation.

To the author's parents for their constant encouragement and

support and to his wife for her inspiration and understanding during the

course of this investigation, this work is dedicated.















TABLE OF CONTENTS


Page
ACKNOWLEDGEMENTS................................................ ii

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

LIST OF APPENDIX TABLES ......................................... viii

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

ABSTRACT ................ ..................................... x

CHAPTER
I INTRODUCTION............... ......................... .

II LITERATURE REVIEW ....................................... 3

Functions of Manganese .............................. 4
Manganese Deficiency................ ............... 6
Rats and mice.................................... 6
Poultry ..................... ..................... 7
Swine.............................................. 9
Ruminants ........................................ 10
Factors Affecting Manganese Requirement............. 11
Manganese Absorption and Excretion................... 13
Blood Clearance and Tissue Deposition of
Manganese ..................... ........ ...... .. 36
Manganese Toxicity.................................. 17

III EXPERIMENT 1. INFLUENCE OF DIETARY MANCANESE LEVEL
ON THE METABOLISM 541n, STABLE MANGANESE AND OTHER
MFNERAL ELEMENTS ................ ................... 22

Experimental Procedure .............................. 23
Results.............................................. 26
Absorption, Excretion, Blood Clearance and
Deposition of 54 N ........... ....... ........... 26
Absorption, Excretion and Blood Levels of
Stable M'antganese ............... .. ............. 36
Apparent Digestion and Tissue DepositJ on of
Dietary Constituent ............................ 39
Discussion... ................. ................. ...... 45
Sumiinry.............................................. 8i








CHAPTER Page
IV EXPERIMENTS 2 AND 3. BIOLOGICAL ASSAY OF INORGANIC
MANGANESE FOR CHICKS.................................. 50

Experimental Procedure............................... 51
Results.............................................. 55
Discussion ........................................... 64
Summary............................................... 65

V EXPERIMENTS 4 AND 5. BIOLOGICAL AVAILABILITY OF SEVERAL
INORGANIC FORMS OF MANGANESE TO CHICKS................ 67

Experimental Procedure............................... 67
Results.............................................. 69
Discussion ........................................... 84
Summary .............................................. 88

VI SUb ARY AND CONCLUSIONS.................................. 90

Absorption, Blood Clearance, Tissue Deposition
and Excretion of Manganese and Other Elements
in Sheep........................................... 90
Biological Assay for Manganese Availability in
the Chick.......................................... 92

APPENDIX.......................................................... 94

BIBLIOGRAPHY ..................................................... 102

BIOGRAPHICAL SKETCH .............................................. 109














LIST OF TABLES


TABLE Page
1 COMPOSITION OF BASAL DIET .............................. 24

2 MAIN AND SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL
AND PATHWAY OF RADIOISOTOPE ADMINISTRATION ON
ACCUMULATED PERCENT OF THE 54Mn DOSE EXCRETED IN
THE FECES............................................ 27

3 MAIN AID SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL
AND PATHWAY OF RADIOISOTOPE ADMINISTRATION ON
ACCUMULATED PERCENT OF THE 541 DOSE EXCRETED IN
THE URINE............................................. 30

4 MAIN AND SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL
A~nI PATHWAY OF RADIOISOTOPE ADMINISTRATION ON
TISSUE RETENTION OF RADIOACTIVITY .................... 34

5 MAIN AK- SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL
AND PATHWAY OF RADIOISOTOPE ADMINISTRATION ON
THE SPECIFIC ACTIVITY IN TISSUE ........ ........ ..... 35

6 MAIN AND SIMPLE EFFECTS OF DIETARY IANG:ANESE LEVEL
AND PATIHWAY OF RADIOISOTOPE ADMINISTRATION ON DAILY
ABSORPTION AND EXCRETION OF STABLE MANGANESE ......... 37

7 MAIN ANDJ SIMPLE EFFECTS OF DIETARY MAGCANESE LEVEL
AND PATHWAY OF RADIOISOTOPE ADMINISTRATION ON
HEMATOCRIT AND PLASMA MANGANESE VALUES............... 38

8 MAIN AND SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL
AND PATlHWAY OF RADIOISOTOPE ADMIINISTRATION ON
DIGESTIBILITY OF ORGANIC LATTER AND DRY LATTER
AND APPARENT ASH ABSORPTION ................. ....... 40

9 MAIN AN'D SIMPLE EFFECTS OF DIETARY MIANGANESE LEVEL
AMD PATilWAY OF RADIOISOTOPE ADMINISTRATION ON
PERCENT APPARENT ABSORPTION OF MINERAL ELEMENTS
BY SHEEP .............................................. 4

10 MAIN AID SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL
AND PATHWAY OF RADIOISOTOPE ADMINISTRATION ON
TISSUE MINE ,,AL CO;IP.OS' LIIO: OF SHEEP .......... ........ 42







TABLE Page
11 CHEMICAL COMPOSITION OF MANGANESE SUPPLEMENTS,
THEIR SOLUBILITY IN VARIOUS SOLVENTS AND X-RAY
PATTE NS ......................................... ...... 52

12 COMPOSTTION OF BASAL DIET ................................ 53

13 BODY WEIGiHTS OF 28-DAY OLD CHICKS FED VARIOUS FORMS
AND LEVELS OF SUPPLEMENTAL MANGANESE (EXPERIMENTS
2 AND 3) ................. ... .................... ...... 56

14 LEG ABNORMALITY SCORES OF 28-DAY OLD CHICKS FED
VARIOUS FORMS AND LEVELS OF SUPPLEMENTAL MANGANESE
(EXPE IENTS 2 AND 3) ................... .................... 58

15 BONE ASH OF 28-DAY OLD CHICKS FED VARIOUS FOIRIS
AND LEVELS OF SUPPLEMENTAL MANGANESEI................ 59

16 TIBIA BREAKING STRENGTH OF 28-DAY OLD CHICKS FED
VARIOUS FORMS AND LEVELS OF SUPPLEMENTAL MANGANESE... 60

17 BONE (TIBIA PLUS FEMUR) MANGANESE LEVELS OF 28-DAY
OLD CHICKS FED VARIOUS FORMS AND LEVELS OF
SUPPLEMENTAL MANGANESE......... ..... ......... ....... 61

18 MANGANESE CONTENT, SOLUBILITY AND X-RAY PATTERNS OF
MANGANESE SUPPLEMENTS ................ ................ 68

19 BODY I\WIGHTS OF 28-DAY OLD CHICKS AS INFLUENCED BY
SOURCE OR LEVEL OF SUPPLEMENTAL MANGANESE............. 7]

20 LEG ABNORMALITY SCORES OF 28-DAY OLD CHICKS AS
INFLUENCED BY SOURCE OR LEVEL OF SUPPLEMENTAL
MANGANESE ............................................... 72

21 PERCENTAGE BONE ASH IN TIBIA AND FEIMUR FROM 28-DAY
OLD CHICKS AS INFLUENCED BY SOURCE OR LEVEL OF
SUPPLEMENTAL MANGANESE ........................... 77

22 BONE (TIBIA PLUS FEMUR) MANGANESE LEVELS OF 28-DAY
OLD CHICKS AS INFLUENCED BY SOURCE OR LEVEL OF
SUPPLEMENTAL MANGANESE.................... ........... .. 78

23 BONE (TIBIA PLUS FEMUR) MANGANESE LEVEL OF 28-DAY
OLD CHICKS AS INFLUENCED BY SOURCE OR LEVEL OF
SUPPLEIENTAL MANCANESE .... .. ...................... 80

24 RELATIVE AVAILABILITY OF MANGANESE TEST SOURCES AT
A SUPPLEMENTAL LEVEL OF 10 PPM ...................... 87














LIST OF APPENDIX TABLES


TABLE Page
25 LIVER LEVELS OF VARIOUS ELEMENTS IN LAMBS FED
DIFFERENT LEVELS OF DIETARY LANGANESE AND DOSED
EITHER ORALLY OR INTRAVENOUSLY WITH 54Mn............. 95

26 KIDNEY LEVELS OF VARIOUS ELEMENTS IN LAZtS FED
DIFFERENT LEVELS OF DIETARY MIANGANESE AND DOSED
EITHER ORALLY OR INTRAVENOUSLY WITH 54,n ............. 96

27 HEART LEVELS OF VARIOUS ELEMENTS IN LIA1 S FED
DIFFERENT LEVELS OF DIETARY M.ANGA-SE AND DOSED
EITHER ORALLY OR INTRAVENOUSLY WITH 5:'ln............. 97

28 SPLEEN LEVELS OF VARIOUS ELEMENTS IN LAUS FED
DIFFERENT LEVELS OF DIETARY MANGANESE AND DOSED
EITHER ORALLY OR INTRAVENOUSLY WITH 54! ............. 98

29 MUSCLE LEVELS OF VARIOUS ELEMENTS IN LAIMS FED
DIFFERENT LEVELS OF DIETARY MAGC-ANFSE A ID DOSED
EITHER ORALLY OR INTRAVENOUSLY WITH 54Mn ............. 99

30 BONE LEVELS OF VARIOUS ELEMENTS IN LAMIBS FED
DIFFERENT LEVELS OF DIETARY IJ!ANGANESf AND DOSED
EITHER ORALLY OR INTRAVENOUSLY WITH 541n............. 100

31 BRAIN LEVELS OF VARIOUS ELErENTS IN LAIDBS FED
DIFFERENT LEVELS OF DIETARY i;;ANGAESE AND DOSED
EITHER ORALLY OR INTRAVENOUSLY WITH 54-............... 101


Vj 1














LIST OF FIGURES


FIGURE Page
1 Accumulated Percent of 54Mn Dose Excreted in
the Feces............................................ 28

2 Accumulated Percent of 54Mn Dose Excreted in
the Urine ............................................ 31

3 Plasma 54Mn Levels of Lambs Fed Different Levels of
Dietary Manganese After IV 54Mn Dose................. 32

4 Relation of Bone (tibia plus femur) Manganese Levels
to Manganese Intake in the Form of Manganese
Sulfate .............................................. 63

5 Representative Tibiae from 28-Day Old Chicks
Raised on Varying Levels of Dietary Manganese
in Experiment 5...................................... 74

6 Representative Tibiae from 28-Day Old Chicks
Raised on a Diet Containing 10 ppm Supplemental
Manganese Supplied from Various Sources in
Experiment 5. Manganese Sources Represented
from Left to Right are: No.'s 2, 4, 6, 8, 12 and 13. 75

7 Relation of Bone (tibia plus femur) Manganese
Levels to Manganese Intake in the Form of
Manganese Sulfate (Experiment 4) ..................... 82

8 Relation of Bone (tibia plus femur) Manganese
Levels to Manganese Intake in the Form of
Manganese Sulfate (Experiment 5) ..................... 83







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

MANGANESE UTILIZATION BY
RUMINANTS AND POULTRY

By

Larry Thomas Watson

December, 1970

Chairman: Dr. C. E. Ammerman
Major Department: Animal Science



Studies were conducted to obtain information on certain nutritional

aspects of manganese in sheep and poultry. The data obtained involved

the effects of different levels and sources of dietary manganese on the

utilization of manganese and other mineral elements within the animal

body. In Experiment 1, lambs receiving a basal diet, containing 30 ppm

manganese, or the basal diet plus 4,000 ppm supplemental manganese were

dosed either orally or intravenously with 54Mn. Limited intestinal

absorption of manganese was demonstrated by low values recorded for

apparent absorption and net retention of stable manganese. In addition,

plasma and tissue uptake of 54Mn by those animals receiving oral doses

of the radioisotope was very low. Excretion of manganese from the body

was almost exclusively by way of the feces regardless of dietary

manganese level or pathway of radioisotope administration. The tissues,

listed in decreasing order of enrichment with radioactive manganese,were

as follows: kidney, liver, spleen, brain, heart, bone and muscle. The

greatest tissue retention of radioactive manganese was demonstrated by

the treaty, nt group fed the basal diet and given the radioisotope intra-

venously. Lambs fed the manganese -supplemented diet had higher levels







of stable manganese in the tissues and plasma than those fed the unsupple-

mented basal diet. The high manganese diet also resulted in decreased

intestinal absorption of iron and phosphorus and decreased liver concen-

trations of iron and zinc.

Experiments 2 and 3 were conducted to develop a suitable biological

assay for manganese availability with chicks. Reagent-grade manganese

sulfate was chosen as the reference standard and added to a semi-purified

basal diet at levels of 10, 20 and 30 ppm manganese. The other test

materials, two commercial feed-grade manganese oxides, were supplemented

at levels of 10 and 20 ppm manganese to the same diet. Ten ppm manganese

from the sulfate or one of the oxide sources were adequate for normal

growth and bone ash and prevented the occurrence of perosis. However,

10 ppm manganese from the other test source were inadequate for normal

responses. Bone manganese levels were found to be more directly related

to dietary manganese levels than the other responses measured. Thus, it

was suggested that bone manganese concentration may be sufficiently

sensitive to serve as a response criterion in a manganese availability

assay when manganese sulfate is used as the standard source.

Experiments 4 and 5 were duplicate trials designed to determine the

availability of manganese from several inorganic sources using the assay

method suggested in Experiments 2 and 3. The reference standard,

manganese sulfate, was supplemented to the basal semi-purified diet at

levels of 10, 20, 30, 60 and 120 ppm manganese. Six other inorganic

manganese sources were tested at a supplemental manganese level of 10 ppm.

Body weight and percentage bone ash cwre not sensitive enough as response

criteria to detect differences in Jiological avai bi lily among source

of Iangenese. The test sources differed in their effectiveness in







preventing perosis. This indicates that incidence of leg abnormalities

is a useful response criterion for manganese availability at dietary

levels of 10-15 ppn manganese. Above this level, however, abnormalities

appear to be prevented entirely.

A highly significant correlation existed between bone manganese

concentration and dietary levels of the element. Bone manganese increased

linearly with increasing dietary manganese, at least up to a dietary level

of 35 ppm total manganese. Thus, it was concluded that tests for biologi-

cal availability of manganese should be conducted at dietary levels below

35 ppm in the area of maximum response. In these experiments, differences

in availability of manganese from test sources were found when bone

manganese levels were expressed as percentages relative to the response

obtained with the standard, manganese sulfate, when supplied at the

same dietary manganese level.













CHAPTER I


INTRODUCTION



Mineral elements constitute only 4 to 6 percent of the

vertebrate body. More specifically, manganese makes up about 0.0003

percent a very small but important amount. Manganese is necessary

for growth, bone formation, and reproduction. However, attempts to

delinate the specific biochemical role or roles of the element have

failed.

Manganese has been known to be a constituent of plant and animal

tissue for more than 50 years. It was first demonstrated to be an

essential element for animals in 1931 (Kemmerer, Elvehjem and Hart).

Nutritional research with manganese gained practical significance in

1936 (Wilgus, Norris and Heuser) and 1937 (Lyons and Insko) when it was

discovered that two commonly occurring diseases in poultry, perosis and

nutritional chondrodystrophy, could be prevented by manganese supple-

mentation.

The manganese content of soils and plants has been demonstrated to

be quite variable. Nutritional disabilities attributed to manganese

deficiency have been reported with grazing cattle under field conditions

in England, the Netherlands (Underwood, 1966; and Bourne, 1967) and

the United States (Dyer, 1961). In other areas, where the manganese

content of soils is relatively high, grazing cattle have shown signs of









antagonistic relationships between manganese and other elements

(Gallup et al., 1952).

Numerous research studies have been conducted concerning the dietary

requirements for manganese. The requirement has been found to be quite

variable, being affected by such factors as the species and breed of

the animal being considered, the age of the animal, the chemical form

of the manganese being offered, and the level of other elements in the

diet which have been shown to be antagonistic to manganese utilization.

The experiments reported herein were conducted to study the effects

of level and source of dietary manganese on metabolism and animal

performance in ruminants and poultry. In Experiment 1 sheep were fed

either high or low levels of dietary manganese and dosed either orally

or intravenously with 54In, a radioisotope of manganese. Measurements

of absorption, excretion, blood clearance and tissue deposition of both

the radioactive and stable isotope of manganese were made. Experiments

2, 3, 4 and 5 were conducted with chicks in an effort to develop a

suitable biological assay for manganese availability. Many commercial

inorganic sources of manganese exist; however, their relative values as

dietary supplements are unknown. These experiments used growth, leg

development, percent bone ash and manganese content of bones as criteria

for availability of the element from several inorganic sources.














CHAPTER II


LITERATURE REVIEW



The biology of manganese suffers from the lack of unifying

principles. A vast accumulation of information and experience exists

concerning manganese metabolism, but no precise function can be

ascribed to the metal in vivo. Since Bertrand (1913) demonstrated the

presence of manganese in plant and animal tissues it has been found to

be essential for normal growth, skeletal development, reproductive

performance and function of the central nervous system. However, its

exact biochemical function or functions remain unknown.

The absence of other unknown but essential nutrients from experi-

mental diets and the trace amounts of manganese required by most animal

species caused early attempts to demonstrate the essentiality of

manganese to be unsuccessful. It was not until 1931 that two groups of

workers independently discovered that manganese is necessary for growth

and fertility in mice and rats (Kemmecrcr, Elvehjem and Hart, 1931; and

Orent and McCollum, 1931).

Manganese first became of practical significance to the nutrition-

ist in 1936 and 1937 when two poultry diseases, perosis or "slipped

tendon" (Wi]gus, Norris and Heuser, 1936 and 1937) and nutritional

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

inadequate intakes of manganese from certain practical diets and could









maintenance of proper amounts of mucopolysacchar.des in epiphyseal

cartilage and bone.

It has been suggested also that manganese serves as activator for

the enzyme arginase by Boyer, Shaw and Phillips (1942) and Wachtel,

Elvehjem and Hart (1943) since a manganese deficiency in rats resulted

in a decreased activity of this enzyme.

Cotzias (1958) postulated that since birds require more manganese

than other species and have a higher body temperature with more oxygen

consumption, maybe manganese is involved in some oxidation-reduction

processes. In 1954, Lindberg and Ernster reported that the catalytic

units of the respiratory chain capable of generating energy-rich

phosphate bonds occur in two forms: (1) a non-phosphorylative form, or

an enzyme-coenzyme complex; or (2) a phosphorylative form containing

adenosine-triphosphate as a cofactor and linked to the non-phosphorylative

form by means of mtanganose. If this is the function of manganese, then

it would necessarily appear in relatively great concentrations within

the mitochondria where the respiratory chain operates. This indeed was

confirmed by 'Iaynard and Cotzias (1955) when they demonstrated that

tissues with the highest uptake (liver and kidney) of intraporitoneally

injected radioactive manganese are also rich in mitochondria. About

one-half of the manganese activity found in these tissues was found to

be located in the mitochondria. Thus, it is possible that the

concentration of manganese in mitochondria is a reflection of its role

as a respiratory cofactor.








be prevented by supplementation of the element to these diets. These

findings led to extensive investigations of dietary requirements and

tissue concentrations of manganese in different animal species.



Functions of Manganese

The specific function or functions of manganese, as stated earlier,

are still unknown. The element, however, has been suggested as functional

in several processes within the body. Since the most pronounced

physical manifestations of manganese deficiency are bone abnormalities,

manganese has been implicated in the calcification or formation of bone.

Wiese et al. (1939) reported that blood and bone phosphatase activity

in chicks with manganese deficiency is lower than normal and suggested

that manganese might serve as an activator for this enzyme. This

finding was duplicated in swine (liebholz, Speer and Hays, 1961), cattle

(Rojas, Dyer and Cassatt, 1965) and sheep (Lassiter and lorton, 1968).

However, manganese deficiency in rats has been shown to have no effect

on phosphatase activity by two groups of researchers (Wachtel, Elvehjem

and Hart, 1943; and Hurley, Everson and Geiger, 1959).

Leach and fluenster (1962) and Leach (1968) found lesions in the

epiphyscal plate before external symptoms of bone weakness appeared in

chicks fed a manganese-deficient diet. They found that osseous tissue

from manganese-deficient chicks had decreased levels of chondroitin

sulfate, and the cpiphyseal plate showed the greatest reduction in this

matrix constituent. Thus, they suggested that manganese is required for

proper formation of the organic matrix of cartilage and hone. Savage

(1968) also supported this view in a review of manganese nutrition in

poultry species stating that manganese probably functions in the









Manganese Deficiency



Rats and Mice

Orent and McCollum (1931) demonstrated that testicular degeneration

occurred in rats raised on a manganese deficient diet. In the same

year, Kemnerer, Elvehjem and Hart (1931) found that female nice raised

on an all-milk diet grew poorly and failed to ovulate normally upon

reaching maturity. When manganese was supplemented in the diet, growth

was stimulated and ovulation was normal.

It was reported by Skinner, Van Donk and Steenbock (1932) that

female rats raised on an all-milk diet fortified with iron and copper

were slow in attaining sexual maturity. However, when manganese was

added to the same diet at a level of about 10 ppm, first estrous was

exhibited at the normal age. In further studies with rats raised on

diets either low or practically devoid of manganese, Boyer, Shaw and

Phillips (1942) found that in manganese-deficient females, estrous

cycles were absent or irregular with a delay in the opening of the

vaginal orifice. The same authors showed that testicular degeneration

and sterility resulted in male rats grown on low-manganese diets. They

also recorded a reduced concentration of liver arginase in manganese-

deficient rats.

Watchel, Elvehjem and Hart (1943) fed a diet supplying five micro-

grams of manganese per day to growing rats. These animals exhibited

an impaired growth rate, which was more pronounced with higher calcium

to phosphorus ratios. They also had poor bone formation, having 3

percent less bone ash than those animals receiving a control diet. In

addition, deficient rats were slightly anemic and had a reduced liver








arginase activity. More recently, Hurley et al. (1961) showed that

feeding a milk diet containing 0.13 ppm manganese to growing rats

resulted in reduced total body length as well as reduced length of long

bones. Tibias were greatly thickened and distorted in shape as compared

to tibias from identical rats receiving the basal milk diet supple-

mented with 6 ppm manganese.



Poultry

Perosis, as described by Gallup and Norris (1939a), is a deficiency

disease affecting the development of bone. The characteristic symptoms

are enlargement of the tibio-metatarsal joint, bending of the distal

end of the tibia and proximal end of the metatarsus and displacement of

the achilles tendon. Locomotion is impaired, and in severe cases the

animal is unable to stand. The outward manifestations of injury give

rise to the descriptive name "slipped tendon".

Titus and Ginn (1931) and Titus (1932) first proposed the name

perosis and stated that rice bran contained a factor that prevents the

disease. Heller and Penquite in 1936 and 1937 reported a protective

factor in a water-soluble extract of rice bran. In that same year,

Sherwood and Fraps (1936) found that the ash of wheat gray shorts was

protective against perosis and thus concluded that the factor was

inorganic in nature.

Wilgus, Norris and Heuser (1936 and 1937) were first to link

perosis with a manganese deficiency. They stated that manganese and

other trace elements were essential for the prevention of perosis in

chickens. Their research showed that the perosis-preventing properties

of cereals are directly related to their manganese content. They fed








a diet containing 10 ppm manganese which resulted in a high incidence

of perosis. However, when 10 to 15 ppm manganese were added to this

diet, growth was stimulated and perosis was prevented entirely. A

supplement of zinc and aluminum seemed essential but was less effective

in preventing the disorder. They found that iron at high levels seemed

to increase the manganese requirement, a result which was later

substantiated. They also found that steamed bone meal as well as pure

calcium phosphate aggravated the occurrence of perosis or increased the

manganese requirement. Insko, Lyons and Martin in 1938 also reported

that manganese is protective against perosis, but contrary to the

results of Wilgus they reported that 30 ppm each of aluminum and zinc

in the ration did not protect but actually increased the incidence of

perosis.

Caskey and Norris (1938) found that a diet containing 15 ppm

manganese, 1.0 percent calcium and 0.5 percent phosphorus was more

effective in preventing perosis than the same basal diet containing

140 ppm manganese, 3.0 percent calcium and 1.5 percent phosphorus. They

postulated that high calcium and phosphorus levels block the absorption

of manganese from the intestinal tract as will be discussed later.

Lyons and Inso- (1937) demonstrated that another nutritional

disease of poultry, termed nutritional chondrodystrophy, could be pre-

vented by manganese supplementation. This is a disease of the unborn

fetus which results in gross skeletal malformations and embryo mortality.

These authors found that supplementation of 40 ppm manganese to the

diet of hens or injection of 0.03 milligrams of the element into the

egg would prevent the disease entirely. Gallup and Norris (1939b)

reported that a layer diet containing 13 ppm manganese resulted in low








egg production and a high incidence of embryo mortality. They also

found that when chicks were hatched from these eggs, they had low

reserves of the element and were susceptible to perosis. The manganese

content of the egg was found to be directly related to the dietary

manganese level of the hen. Hill and Mathers (1968) reported that the

dry matter of eggs from pullets receiving 6.5 or 50 ppm dietary manganese

contained 0.66 and 1.50 ppm of the element, respectively. In 1968,

Savage demonstrated that manganese is required at dietary levels of

from 54 to 108 ppm in turkeys for normal egg production and hatchability.



Swine

Killer et al. (1940) reported that corn-soy type diets containing

11-14 ppm manganese caused lameness in 50 percent of the pigs receiving

such diets. They found that manganese sulfate added at levels of 50

to 60 ppm manganese prevented the lameness but were ineffective in

curing stiffness after it had developed. Johnson (1943 and 1944)

demonstrated satisfactory growth of pigs receiving natural feedstuffs

containing 7-10 ppm manganese from weaning to market weight. Repro-

duction was normal through two generations in pigs raised on these

feedstuffs. However, when pigs were raised on a diet containing 0.5

ppm manganese they failed to reproduce normally. Reproduction was

satisfactory when the manganese level of this diet was raised to 6

ppm. Grummer et al. (1950) showed that growth of pigs confined to a

concrete feeding floor and receiving a corn-soy ration containing 12

ppm manganese could be improved by the addition of 40 ppm manganese to

the basal diet. In contrast, Liebholtz, Speer and Hays (1961) found no

growth depression or adverse feed utilization by pigs fed a diet








containing only 0.35 ppm of manganese. However, they found a decrease

in bone manganese and alkaline phosphatase activity.

Plumlee et al. (1956) also found no difference in rate of gain or

feed efficiency over the growth period of pigs fed a semi-purified diet

containing from 0.5 to 40 ppm manganese. Boars raised on a dietary

level of 3.3 ppm manganese grew normally and showed normal spermato-

genesis. However, gilts fed through growth, gestation and lactation on

a diet containing 0.5 ppm manganese exhibited reduced skeletal growth

and weakness, irregular estrus cycles, fetal resorption, low milk

production and decreased tissue levels of manganese.



Puminants

Bentley and Phillips (1951), in experiments with the effects of

low dietary manganese on dairy cattle, reported that 10 ppm manganese

in the diet were adequate for growth of heifers since higher levels did

not stimulate faster growth. However, the heifers receiving only 10

ppm manganese were slower to exhibit their first estrous, were slower to

conceive upon breeding, and gave birth to a higher percentage of calves

with leg deformities than did control heifers raised on diets containing

30 ppm manganese. The cattle were slaughtered after three lactations

with post-mortem examination and analysis revealing no differences in

tissue concentration of manganese except in the ovaries, but revealing

abnormal structural changes in the liver of those animals receiving the

lower manganese level in the diet. They concluded that 20 ppm

manganese in the diet seemed adequate for growth and reproduction under

normal conditions. However, Dyer, Cassatt and Rao (1964) reported that

23 ppm manganese were inadequate for gestating beef heifers, resulting









in leg deformities in the newborn and reduced liver and bone concentra-

tions of manganese in the dam. They found 61 ppm to be the minimum

level of manganese which resulted in normal calves with normal tissue

concentrations of the element. Dyer and Rojas (1965) reported that 45

ppm seemed adequate for reproduction. In continued work with manganese

deficiency in cattle, Rojas, Dyer and Cassatt (1965) found that 16 ppm

or less of manganese were inadequate for reproduction resulting in

calves with neonatal deformities with reduced breaking strength and

length of the humerus, reduced serum alkaline phosphatase activity, and

lower manganese levels of bone, liver, and gonads. They suggested that

about 20 ppm of manganese would be adequate for reproduction, but that

ingestion of compounds antagonistic to manganese utilization may increase

the dietary requirement of the element.

In recent work by Lassiter and Morton (1968), effects of low

manganese diets fed to lambs were studied. Four sets of twin ewe lambs

were used, one from each set receiving 0.8 ppm of manganese in a puri-

fied diet, the other receiving 29.9 ppm in the same diet. After 16

weeks those receiving low dietary manganese exhibited weak joints, a

decreased feed intake, and a reluctance to move. The lambs were

slaughtered at 22 weeks with post-mortem examination showing that those

fed the low manganese diet had shorter tibias with less breaking

strength and a reduced ash content.



Factors Affecting Manganese Requirement

The requirements for mngonese are quite variable and are affected

by the criLcrin of adequacy employed, the chemical form, the nature of

the diet, and Ilie breed and species of the animal under consideration.








The amount of manganese required for reproduction and other stress

conditions is higher than that for growth and maintenance. Bentley and

Phillips (1951) found, and their results were substantiated by Garrett

(1964), that 9 to 10 ppm of manganese were adequate for growth of

calves. However, they found this amount to be entirely inadequate for

normal sexual maturity to be attained. Therefore, a level of dietary

manganese may be high enough for growth, but inadequate for reproduction.

Differences in biological availability of manganese from different

sources or chemical forms can also alter the animal's requirement for

the element. Schaible, Bandemer and Dividson (1938) reported that

practically all chemical forms of manganese are of equal value to the

animal except for a few carbonate and silicate ores. They stated that

animals receiving diets high in corn require supplemental manganese

since corn, as is true for many small grains, has a very lo: manganese

content. They reported that manganese content of grain and forage

plants varies according to culling time, stage of maturity, fertili-

zation and soil reaction. Soil reaction seemed to be the most important

factor with manganese being much higher in plants grown on acid soils.

Gallup and Norris (1939a), working with manganese requirements for

chicks, found that manganese chloride, sulfate, carbonate, and oxide

were of equal value in preventing perosis when supplemented at a level

of 50 ppm manganese to a basal diet containing 10 ppm of the elme nt

naturally. Combs (1951) also stated that the chemical forms: manganese

sulfate, manganese chloride, manganese carbonate, manganese dioxide,

potassium permanganate, and the manganese ores: manganite, pyrolusite,

hausmannite and hlemaite serve as satisfactory sources of manganese for

the chick. He found that a manganese oxide ore was 85 percent as









effective as manganese suliate in preventing perosis when supplemented

at dietary levels of 15 or 25 ppm manganese but was of equal value when

added at levels of 35 and 55 ppm.

Hennig et al. (1967) more recently compared the manganese uptake

of broilers from various radioactive compounds including the sulfate,

chloride and oxide forms of manganese. They found that radioactive

manganese as manganous chloride was incorporated into the body to a

significantly larger extent than that from the other compounds tested.

The requirement for manganese is quite variable among species as

discussed earlier, with poultry species having a higher dietary

requirement than most others. Differences have also been reported in

the manganese requirements of breeds and strains within species as

demonstrated in chickens by Gallup and Norris (1939a). In one strain

of the New Hampshire breed, 50 ppm of supplemental manganese added to a

basal diet containing 10 ppm of manganese reduced the occurrence of

perosis from 80 percent to 4 percent while in another strain which

seemed more susceptible to the disease, the same level of supplemental

manganese only reduced the level of perosis to 18 percent. They found

also that 30 ppm of supplemental manganese were as protective against

perosis in the White Leghorn breed as were 50 ppm in the New Hampshire

breed.



Manganese Absorption and Excretion

Dietary requirements for manganese seem quite high in comparison

to the trace concentrations of the element within the body. This is

due to poor absorption of manganese from the gut and the antagonistic

relationship which exists between manganese and other elements and

compounds rcsentn in the diet or intestinal tract.









It was stated by von Oettingen (1935) that dietary manganese is

slowly and incompletely absorbed from the intestinal tract, varying as

to the acidity of the intestinal environment and the solubility of

manganese compounds. He said further that manganese is excreted pri-

marily in the colon and bile and only moderately in the urine. Skinner,

Peterson and Steenbhck (1931) reported that adult rats excreted from

80 to 99 percent of their dietary manganese in the feces depending on

the level of manganese in the diet; more is excreted when an excess is

present.

Greenburg, Copp and Cuthbertson (1943) found about 30 percent of

an intraperitoneal injection of a radioactive manganese dose appearing

in the bile within 48 hours. They stated that 50 to 75 percent of the

injected dose appearing in the feces was carried by the bile. According

to Cotzias (1958), manganese is absorbed in the rat at a level of 3 to

4 percent with excretion almost exclusively in the feces and significant

excretion in urine only when abundant chelates are present. He also

reported that manganese excreted into the bile or gut is partially

reabsorbed. In a later paper Cotzias (1960) stated that excretion of

manganese in bile is directly related to dietary intake.

Koshida, Kato and Hara (1963) and Kato (1963) presented evidence

of manganese excretion through the intestinal epithelium. After the

injection of a radioactive isotope of manganese they found conspicuous

radioactivity in the intestinal epithelium and also in the intestinal

mucus cohering to free surface. Papavasiliou, miller and Cotzias (1966)

reported that bile duct obstruction only diminished excretion of a

radioactive done of manganese. They stated that bile formation

constitutes the main regulation of manganese excretion but, if over-

loaded, other excretion routes participate.









Brown and McCracken (1965) conducted a manganese balance trial

with chickens using the isotope dilution technique. They found that

when feeding a diet containing 15 ppm manganese to laying pullets, the

apparent absorption of manganese was about 30 percent. Manganese

retention was 1.6 milligrams per day which is high relative to the

birds' requirement. Lassiter (1966) and Lassiter and White (1966)

published results of manganese balance trials with sheep revealing that

net absorption of the element varies between 8 and 19 percent, depending

on the level of mineral supplementation in the diet used. They also

reported that there was no detectable excretion of manganese in the

urine.

Wilgus, Norris and Heuser (1936) found that the perosis-prcventing

properties of a diet were related to its manganese content as has beer

stated earlier. They also reported that excess calcium and 'phosphorus

aggravated the condition. In 1939, Uilgus and Patton reported that

excess calcium was essential for the stimulation of perosis. They

found that calcium phosphate in the gut precipitated manganese ions

from solution, and that feeding calcium phosphate as steamed bone meal

decreased diffusible manganese in intestinal contents, thereby decreas-

ing absorption and increasing the dietary manganese requirement. They

also discovered that ferric hydroxide or ferric citrate had a similar

effect on manganese if sodium chloride were present. Bourne (1967)

stated that work with cattle in England also indicates that high levels

of calcium and phosphorus in the diet increase the dietary manganese

requirement by interfering with absorption of the element from the

intestinal tract.








Blood Clearance and Tissue Deposition of Manganese

Borg and Cotzias (1958a and 1958b) and Cotzias (1963) have reported

blood clearance values for rats and humans after intravenous injections

of the radioisotopes of manganese, 54 and 56Mn. They recorded a

rapid disappearance of the element from the plasma. Within 10 minutes

less than 10 percent of the dose remained in the plasma, and after 70

minutes radioactivity was only barely detectable in the plasma.

Activity began to reappear in circulating red blood cells after 36 to

48 hours. They stated that most or all endogenous manganese is avail-

able for interchange within an hour and concluded that manganese must

exist in the body in dissociable chelates or other relatively labile

intracellular combinations.

It has been reported in swine (Plumlee et al., 1956), rats (Cotzias,

1963), and chickens (Settle et al., 1969) that after administration of

radioactive isotopes of manganese, 54n and 56Mn, the relative enrich-

ment of the tissues with radioactivity listed in decreasing order was

as follows: liver, kidney, spleen, heart, bone, muscle and brain. Kato

(1963) using mice, and Rojas, Dyer and Cassatt (1966), using rats, found

similar tissue responses to injection of 52Mn and 56Mn with one

exception. They found that the kidney was more greatly enriched with

radioactivity than the liver. The above researchers as well as Mathers

and Hill (1967) found an inverse relationship between the dietary

manganese level and the percent of the radioactive manganese dose

retained by the various tissues. In contrast, Britton and Cotzias

(1966) reported that the tissue concentration of 54Mn, following an

injection, was directly related to the level of stable dietary manganese.









Thacker, Alderman and Bratton (1956) and Underwood (1962) have

listed normal plasma levels of stable manganese to lie between 0.05 and

0.20 ppm. Results of tissue analyses in rats (Skinner, Peterson and

Steenbock, 1931), swine (Johnson, 1943: and Svajgr, Peo and Vipperman,

1969), chickens (Mathers and Hill, 1968) and cattle (Bentley and Phillips,

1951; and Rojas, Dyer and Cassatt, 1965) are very similar indicating

that manganese concentration of tissue varies from about 0.5 to 12.0 ppm.

Tissues listed in decreasing order with respect co manganese concentra-

tion were: liver, kidney, pancreas, heart, bone and muscle. These

authors also state that tissue concentrations of manganese are directly

related to the dietary level of the element.



Manganese Toxicity

The antagonistic relationship which exists between manganese and

other elements and the wide variation in the level of manganese in

common feedstuffs has stimulated the investigation of effects of high

levels of dietary manganese. Becker and McCollum (1938) fed rats a

diet containing manganese as manganese chloride at levels of 1,800,

3,600, 9,000, 18,000 and 36,000 ppm. Reproduction was normal at all

levels of intake and growth was normal except at the 36,000 ppm level,

at which level some growth depression was recorded. In 1942, Chornock,

Guerrant and Dictcher reported that high dietary levels of manganese

adversely affected growth of rats. They found that when manganese

constituted 1.73 percent of the diet that calcium and phosphorus

excretion in the feces was increased. There was a more pronounced

interference with phosphorus retention than with calcium and an increase

in dietary phosphorus improved its retention.









Gallup et al. (1952) investigated a condition in southeastern

Oklahoma where cattle grazing a native grass in summer and fed native

grass hay plus a protein supplement in the winter developed phosphorus

deficiency unless phosphorus was supplemented. Cattle in north-central

Oklahoma received the same or similar treatEent but no phosphorus

supplement was required. Analysis showed that the grasses in southeast

Oklahoma had slightly less phosphorus hut five to ten times as much

manganese. They then conducted a mineral balance study in which the

diet supplied 7.7 and 14.5 grams of calcium and phosphorus daily.

Manganese was fed at graded levels of 0, 250, 1,000 and 2,000 ppm as

manganous sulfate. It was found that phosphorus excretion in the forces

was increased at all levels of manganese supplemo.ntation greater then

zero. Calcium excretion was increased at 1,000 and 2,000 ppm nanganese

and calcium and phosphorus balances were positive as long as the

manganese content of the diet was 500 ppm or less.

Working with la-,bs, Hartman, Matrone and Wise (1955) investigated

manganese-- ron interrelationships. After depletion of their body iron

stores, lambs were fed a milk diet supplemented with iron, copper,

cobalt and vitamins. Feeding graded levels of manganese in this diet

showed that above 45 ppm of supplemental manganese, hemoglobin concentra-

tion and serum iron decreased, furthermore, levels approaching 5,000 ppm

manganese caused a decreased tissue concentration of iron. In a second

experiuiant in which anemic lambs vtre fed a roughage diet plus 0, 1,000

or 2,000 ppm added manganese, these workers found similar results and

suggested that excess rP'nganese interferes with iron absorption by

antagonizing enzynre systems which o:idize or reduce iron at the

absorption site. 11ansard et al. (1960) supported this statement in an








experiment using rats receiving various levels of manganese in their

diets. They reported that 500 or 1,000 ppm manganese depressed growth

and decreased iron absorption.

In 1959, Matrone, Hartman and Clawson tested the influence of high

levels of manganese on iron metabolism in anemic rabbits and pigs.

They found that when a diet containing 2,000 ppm manganese was fed,

hemoglobin formation was depressed in both pigs and rabbits. The

minimum level of manganese interfering with hemoglobin formation seemed

to be between 50 and 125 ppm. In addition, they found that a supple-

ment of 400 ppm iron overcame the depressing effect of 2,000 ppm

manganese. They also noted growth depression at levels of 1,250 and

2,000 ppm manganese.

Gubler et al. (1954) conducted an experiment concerned with the

influence of manganese on copper metabolism in the rat. They found that

four percent of manganese chloride supplemented to a basal diet decreased

the copper concentration in the kidney and that a microcytic hypochromic

anemia resulted. When a supplement of 0.1 percent copper sulfate was

added to the diet, there was an increase in copper concentration of the

kidney but the anemia still existed. It was suggested by these workers

that manganese forms a complex with copper making it unavailable or

blocks the action of copper containing enzyme systems involved in

erytlropoiesis, thereby causing the anemic condition. Bunch et al.

(1963) found no effect of graded levels of 0, 1,000 and 2,000 ppm

manganese on performance of pigs receiving a diet containing up to 250

ppm copper. In a similar experiment published in 1964 by these workers,

these results were duplicated.








In work with the interaction of manganese and other mineral elements

in ruminants, Pfander, Beck and Preston (1966) found that cobalt uptake

by rumen mi.croorgani-mis was depressed by high levels of dietary

manganese and thai high levels of cobalt plus manganese combined caused

a reduction in the availability of zinc.

In recent years attempts have been made to study the practical

aspects of feeding high levels of manganese to farm animals since there

are several places in the world where high soil and plant concentrations

of manganese exist. Liebholz et al. (1961) conducted experiments

studying the effects of levels of manganese varying from 0 to 4,000 ppm

on baby pig performance and tissue concentrations of the element. In

no case did they find growth depression or adverse feed utilizat-ion due

to high or low manganese levels. However, an increased concentration

of manganese in liver, boeie and hair was noLed at levels of manganese

above 40 ppm.

In 1960, Robinson et al. tested the manganese tolerance of feedlot

cattle receiving 0, 250, 500 and 1,000 ppm manganese in a corn-soy basal

diet. They found no significant differences in average daily gain or

feed efficiency and no effect on hemoglobin, hematocrit or in concentra-

tions of iron, calcium or phosphorus in the blood. They did find,

however, a decrease in iron absorption and a decrease in cellulose

digestion at high levels of manganese. This same group, Robinson et al

(1961), found similar results with growing calves receiving a bermuda-

grass hay diet supplemented with 0, 300 and 600 ppm nanganese. Again

they found no differences in growth, hemoglobin, hematocrit or blood

levels of calcium, phosphorus, nagncsium; or iron. Iron absorption

however was decreased and iron depletion increased at the highest

levels of rmnganese.








Cunningham, Wise and Barrick (1962) conducted an experiment with

growing Holstein calves receiving a corn-soy basal diet supplemented

with 0, 818, 2,455 and 4,911 ppm manganese. They found that average

daily gain, feed intake and feed efficiency were adversely affected by

the two higher levels of manganese supplementation. There were no

differences in hemoglobin concentration or in serum calcium, phosphorus,

magnesium or alkaline phosphatase activity. However, in a similar

experiment where feed intake was held constant for all lots receiving

high or low manganese the results of the first experiment were not

duplicated. They found no significant differences in growth or feed

efficiency but found a decreased hemoglobin concentration in those

animals on high manganese intakes. The results of the second experi-

ment indicated that decreased growth under conditions of high manganese

intake may be largely due to decreased feed intake. A limited study

conducted by these workers with rumen volatile fatty acid production

indicated that production of volatile fatty acids, especially propionic,

is reduced under conditions of high manganese intake indicating a change

in the microbial population of the rumen. Mleghal and Nath (1964) also

noted a change in the population of microorganisms in the ceacum of

rats fed dietary manganese levels of 140 ppm as compared to rats fed

only the basal diet with no supplemental manganese.














CHAPTER III


EXPERIMENT 1. INFLUENCE OF DIETARY MANG.AESE LEVEL ON THE
METABOLISM OF 54Zn, STABLE MANGANESE AND
OTIER MINERAL ELEMENTS



The extremely variable levels of manganese in soils and common

forages indicate the importance of investigating the effects of varying

dietary manganese levels for ruminants. Increasing levels of nanganese

for ruminants have been reported to result in decreased growth, feed

intake and feed efficiency (Cunningham, Wise and Barrick, 1962). It has

also been demonstrated that increasing dietary manganese levels cause

decreases in absorption of iron (Hartnan, Matrone and Vise, 1955; and

Robinson et al., 1960), calcium and phosphorus (Gallup et al., 1952)

from the intestinal tract. There are very few reports, however, involving

the effects of varying manganese levels in the diet upon the metabolism

of manganese itself.

The object of this trial was to investigate the metabolism and

distribution of 54Mn, a radioactive isotope of manganese, and stable

manganese as influenced by dietary intake cf manganese and pathway of

radioisotope administration in lambs. Nutritional interrelationships of

manganese with other mineral elements were also studied.









Experimental Procedure

Sixteen Florida Native wetlier lambs with an average body weight of

34 kilograms were randomly allotted to a 2 x 2 factorially designed trial.

One hundred microcuries of 5411In, a radioactive isotope of manganese with

a half-life of 303 days, as manganese chloride was administered either

orally or intravenously into lambs being fed the basal diet shown in

Table I containing 30 ppm manganese naturally or receiving the basal

diet plus 4,000 ppm added manganese supplied from reagent grade manganese

carbonate supplemented at the expense of corn starch.

The animals were housed in raised metabolism crates from the begin-

ning of the trial and fed 900 grams of their respective experimental

diet daily. Refused feed was weighed back each day prior to feeding,

and tap water containing 0.05 ppm manganese was provided ad libitum

throughout the experiment.

A 21-day preliminary feeding period was followed by the administra-

tion of the respective 54Mn dose given either orally in a gelatin capsule

or injected with physiological saline into the jugular vein as described

by Hansard, Comar and Plumlce (1951). Total fecal and urinary collections

were then made at 24-hour intervals for a period of 21 days. Urine was

collected in plastic buckets to which had been added 100 milliliters of

a solution containing 25 percent hydrochloric acid. Feces were collected

in canvas fecal collection bags.

After completing the collection, a period of 35 days elapsed to allow

for clearance of the radioisotope from the body, after which the lambs

were again dosed with 5111,fn as before. Blood samples were obtained at

periodic intervals for a period of 96 hours by means of a polyvinyl

catheter in the jugular vein as described by HIansard, Comar and Plumlee












TABLE 1. COMPOSITION OF BASAL DIET




Ingredient Percent

Bermudagrass hay1 10.00
Soybean meal (50% protein) 14.00
2
Snapped corn 71.45
Corn starch 1.00
Salt 0.50
Trace mineral mixture3 0.05
Bifos (21% phosphorus, 16% calcium) 1.00
Corn oil 2.00
Vitamins A, D and E5 I
100.00

Hay ground through 3/8 inch (1.6 cm.) screen in hammer mill.
2
Snapped corn with cobs and shucks.

Ingredients % of Mixture Mineral cdd-d to diet, ppm
Iron (FeSO4) 54.50 100.00
Copper (CuC12) 4.20 10.08
Cobalt (CoCO3) 1.01 2.47
Zinc (ZnCO3) 19.20 49.92
Iodine (KI) 1.41 5.43
Corn Starch 19.68
100.00
4
Stabilized with Santoquin at a level of 56 ml. per 5 gallons or 0.0297%
of total diet.
The following vitamins were added per pound of diet:
2000 IU Vitamin A palmitate
270 IU Vitamin D2
5 mg. DL alpha tocopherol








(1951). All animals were then killed and selected tissues, including

liver, spleen, heart, kidney, brain, bone and muscle, removed for

analysis.

Daily fecal and urinary samples as well as tissue and plasma samples

were assayed for 54i n content in a sodium iodide crystal well counter

and a large capacity A-pi scintillation counter (arm counter). Samples

were prepared especially for radioactivity determinations in order to

maintain consistent geometry within the counter. Samples of fresh feces

in the amount of 400 grams were taken daily from the fecal collection

from each animal and compressed to a constant volume. Of the measured

volume of urine excreted daily by each animal, one liter was saved and

if urinary excretion was less than one liter, then it vas diluted to this

volume. Blood samples were centrifuged and four milliliters of plasma

saved for determination of radioactivity. Tissue samples were thoroughly

washed in tap water before determinations of 541In content.

After radioactivity determinations, the pooled fecal collection

from the last seven days of the collection period for each animal was

dried in a forced-air drying oven at 700 Centrigrade. The feces were then

allowed to equilibrate with room air moisture, weighed and a randomly

selected aliquot saved for chemical analysis. Urine samples were saved

in proportion to the daily urinary excretion for each animal from the

last seven days of the collection period. Each sample was then stirred,

filtered through Whatman No. 42 filter paper and approximately 100

milliliters kept for chemical analysis.

Dry matter and ash determinations were made on duplicate feed and

feces samples according to the method outlined by A.O.A.C. (1960).

Determinations for manganese, iron, copper, zinc, calcium, and magnesium








in feed, feces and tissue, and for manganese in urine and plasma were

made by atomic absorption spectrophotometry according to the methods

recommended by the manufacturer (Anonymous, 1964). Phosphorus content

of feed, feces and tissue was determined by the colorimetric method out-

lined by Fiske and Subbarow (1925). The data were analyzed statistically

by analysis of variance for factorial experiments with single degree of

freedom comparisons (Steel and Torrie, 1960).



Results

Absorption, Excretion, Blood Clearance and Deposition of 541Mn

Table 2 shows the accumulated percent of the 54Mn dose excreted by

way of the feces on the seventh, fourteenth and twenty-first days after

dosing. Dietary manganese level and pathway of isotope administration

both had significant (P < .01 and P < .05, respectively) effects on the

percent of the dose excreted in the feces by the seventh day. Those

lambs fed only the basal diet had 77.9 percent of the dose appearing in

the feces as compared to 89.2 percent for those fed supplemental

manganese. Intravenous dosing also resulted in less excretion of the

radioisotope by the seventh day than oral dosing, 79.3 and 87.7 percent,

respectively. Pathway of isotope administration ceased to have a signi-

ficant effect upon the percent of the 541Mn dose excreted in the feces by

the fourteenth day after dosing. However, dietary manganese level

continued to exert a significant (P < .05) effect until the twenty-first

day.

Figure 1 shows graphically the accumulated percent of the 541Mn dose

excreted in the feces with time. Even though dictary manganese level had

a significant effect on the accumulated fecal excretion of 54Mn over the













TABLE 2. MAIN AND SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL AND
PATHWAY OF RADIOISOTOPE ADMINISTRATION ON
ACCUMULATED PERCENT OF THE 54Mn DOSE
EXCRETED IN THE FECES

Dietary Isotope
Manganese Administration 7 14 21
Level Pathway Days Days Days

Main Effects Percent

Low 77.9a 82.2x 83.7x
High -89.2b 90.1y 90.3y
IV 79.3x 83.5 85.1
Oral 87.7y 88.8 89.0

Simple Effects2

Low IV 70.0a 77.5x 80.3x
Low Oral 85.9b 87.0 87.2X''
High IV 88.8b 89.6y 89.9y
High Oral 89.6b 90.7y 90.8y


1Values listed are means of 8 observations.

Values listed are means of 4 observations.

ab Means within thee me column and group with different superscripts
are significantly (P < .01) different.

y Means within the same column and group with different superscripts
are significantly (P < .05) different.













100 '7 '











S60 /
80 / /









60

40 High Mn, Oral ~ :~"

SHigh n.h IV ........V

Low Mn, Oral *

20
Low lin, IV *





7 14 21

Days rollo:ing Dosing

Figure 3. Accumulated Percent of 541n Dose Excreted in the Feces.









entire 21-day collection period, the level of significance decreased with

time. The simple effects shown on this graph (Figure 1) and in Table 2

demonstrate that lambs fed the low manganese diet and given the intraven-

ous radioisotope dose were significantly lower than all other groups in

accumulated fecal excretion of the nuclide early in the collection

period. However, by day 21 they were not significantly lower than those

fed the low manganese diet and given the oral dose.

The accumulated percent of the 54Mn dose excreted in the urine by

the seventh and twenty-first days after dosing is shown in Table 3.

Urinary 54,n accounted for only a very small portion of the total

excretion of the isotope, always less than 1 percent of the daily

excretion. Level of dietary manganese did not significantly effect

urinary excretion of the radioisotope. Pathway of isotope administration,

however, did have a significant (P < .01) effect throughout the entire

collection period. Values of 0.29 and 0.32 percent at 7 and 21 days

were recorded for those given the intravenous dose compared to 0.04 and 0.06

percent for those receiving the oral dose.

The percent of the 54in dose excreted in the urine with time is

depicted graphically in Figure 2. As stated earlier intravenous dosing

resulted in a significantly higher percent of the dose appearing in the

urine regardless of dietary manganese level. Even though there was no

statistically significant effect of the manganese level in the diet,

those animals receiving the high dietary level had consistently more

urinary 54In excretion than animals dosed by the same pathway but fed

the lower dietary level of manganese.

Figure 3 shows graphically the percent of the 54Mn dose remaining

in the plasma of those lambs receiving the intravenous injection of 100











TABLE 3. MAIN AND SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL AND
PATHWAY OF RADIOISOTOPE ADMINISTRATION ON
ACCUMULATED PERCENT OF THE 54Mn DOSE
EXCRETED IN THE URINE



Dietary Isotope
Manganese Administration 7 21
Level Pathway Days Days

Main Effects1

Low 0.14 0.17
High 0.18 0.21
IV 0.29a 0.32a
Oral 0.04b 0.06b

Simple Effects2

Low IV 0.25a 0.29a
Low Oral 0.03b 0.05b
High IV 0.32a 0.35a
High Oral 0.04b 0.06b


Values listed are means of 8 observations.

Values listed are means of 4 observations.

a,b Means in the same column and group with different superscripts are
significantly (P < .01) different.
















0.5




High Mn, IV ...


0.4 Low Mn, IV



. ...... . ..""""""""".
S .......... ..................

S0.3



o


o 0.2
0 /
S* High Mn, Oral


Low Mn, Oral


0.1








7 14 21
Days Following Dosing


Figure 2. Accumulated Percent of 541mn Dose Excreted in the Urine.























High Dietary Mn X---X


Low Dietary Mn o--0


I0







8






d6
ci



0







43
ci

ci
U
a


2






0-
2 4 6 8 10
Hours After 54M1in Dose



Figure 3. Plasna 54Mn Levels of Lambs Fed Different Levels of Dietary
Manganese After IV 54Mn Dose.


II
09






ox
0
0\


a5PC-i~XP~n~B~Cb~L~Z~f~F1-nt~-CBPi~FE









microcuries from time zero to ten hours after dosing. The portion of

the dose retained in the plasma decreased very rapidly to less than 1

percent. This graph indicates that lambs fed high levels of dietary

manganese retained the radioactivity longer than those fed the low

manganese basal diet. However, variation was quite high within groups

and there were no significant differences in percent of dose retained at

any point along the two lines shown. Measurable quantities of the

intravenous 541n dose remained in the plasma for 12 hours after dosing.

However, from 12 to 96 hours, only traces of activity remained. Radio-

activity in the plasma of those lambs given the oral dose of 100 micro-

curies of 54Mn was never detected in more than trace quantities.

Treatment effects on tissue retention of radioactivity 96 hours

after dosing are shown in Table 4. There was a highly significant

(P < .01) manganese-level x pathway-of-isotope-administration interaction

for all tissues analyzed therefore only simple effects or individual

treatment comparisons will be discussed. The average percent of the dose

retained in all tissues tested was significantly (P < .01) greater for

those lambs fed the low manganese basal diet and given the intravenous

dose of the radioisotope. Retention values for this group ranged from

a high of 49.9 percent retained per kilogram of dry tissue in the kidney

to 1.62 percent in the muscle. There were no significant differences

among the other three treatment groups in the percent of dose retained

in the kidney, liver, spleen, heart or muscle. However, those animals

fed the high manganese diet and given the intravenous 54Mn dose exhibited

intermediate retention values in the brain and bone.

Tissue levels of radioactivity are expressed as specific activity,

or counts per minute per nicrogram of stable manganese, in Table 5.


















in (0 (0 I

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There was again a high) significant manganese-level x pathway-of-isotope-

administration interaction, therefore, only individual treatment compari-

sons will be discussed. Numeric trends were very similar, and identical

in most cases, to those for percent of dose retained per kilogram of dry

tissue (Table 4). Single degree of freedom treatment comparisons revealed

that the combination of low dietary manganese and an intravenous radio-

isotope dose resulted in a significantly (P < .01) greater specific

activity than that obtained with all other treatments for any tissue

tested. There were no significant differences in specific activity

among the other three treatments for any tissue.


Absorption, Excretion and Blood Levels of Stable Manganese

The data for dietary intake, fecal and urinary excretion of manganese

and the calculated values for apparent absorption and net retention of

the element are summarized in Table 6. An increase in intake of manganese

from 21.44 to 3,393.36 milligrams per day resulted in significant

(P < .01) increases in fecal and urinary excretion of the clement from

22.85 and 0.35 milligrams to 3,573.93 and 1.47 milligrams, respectively.

There were no significant differences in apparent absorption or net

retention due to treatment with average values for absorption and

retention being negative in every case.

Plasma levels of manganese (Table 7) were significantly (P < .01)

higher for those lambs fed the higher levels of dietary manganese than

those fed only the basal diet, with an average value of 22.24 and 16.14

micrograms per 100 milliliters, respectively. There was no significant

treatment effect on hematocrit values (Table 7), the average for all

animals being 45.07 percent.

















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TABLE 7. MAIN AND SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL AND
PATHWAY OF RADIOISOTOPE ADMINISTRATION ON
HEMATOCRIT AND PLASMA MANGANESE VALUES

Dietary Isotope Plasma
Manganese Administration Hematocrit Manganese
Level Pathway (%) (Ag %)

Main Effects1
Low 44.25 16.14a
High 46.03 22.24b
IV 43.75 20.05
Oral 46.46 18.32

Simple Effects2
Low IV 43.42 16.36a
Low Oral 45.08 15.91a
High IV 44.08 23.75b
High Oral 47.50 20.74b



Values listed are means of 8 observations and means within the same
column and group with different superscripts are significantly
(P < .03) different.
2Values listed are means of 4 observations and means within the same
column and group with different superscripts are significantly
(P < .01) different.








Apparent Digestion and Tissue Deposition of Dietary Constituents

Average coefficients for apparent digestion of dry matter and

organic matter and for apparent absorption of ash or total dietary

minerals are shown in Table 8. Digestion of organic matter and dry

matter was not significantly affected by treatment. The apparent absorp-

tion of ash was significantly (P < .01) higher for those lambs fed the

low manganese diet averaging 47.7 percent compared to an average of 37.5

percent for those receiving supplemental manganese.

Apparent absorption coefficients for iron, copper, zinc, magnesium,

calcium and phosphorus are listed in Table 9. Absorption of copper,

zinc and calcium was not affected by treatment. Low levels of dietary

manganese resulted in significantly (P < .01) more absorption of iron

and phosphorus than the higher dietary level of manganese. Average

absorption values of -3.8 and 33.8 were recorded for iron and phosphorus

respectively when only the basal diet was fed as compared to -47.7 and

17.2 percent absorption when supplemental manganese was supplied. There

was a significant (P < .01) manganese level x pathway-of-radioisotope-

administration interaction effect on apparent absorption of magnesium.

Analyses of simple effects revealed that those lambs fed the low

manganese diet and given the intravenous radioisotope dose exhibited a

significantly (P < .01) lower absorption of magnesium than any other

treatment group. There were no significant differences in magnesium

absorption among the other three treatment groups.

Mean values for the mineral composition of the liver, kidney, heart,

spleen, muscle, bone and brain are shown in Table 10, and values for

individual labs are shown in appendix Tables 25-31. The manganese lefel

in all tissues tested was directly related to the .evel of dietary













TABLE 8. MAIN AND SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL AND PATHWAY
OF RADIOISOTOPE ADMINISTRATION ON DIGESTIBILITY OF ORGANIC
MATTER AND DRY MATTER AND APPARENT ASH ABSORPTION


Isotope
Administration
Pathway


Digestion Apparent
Coefficients (%) Absorption (%)
Organic Matter Dry Matter Ash


Main Effects1


High


Low
High
High


IV
Oral

Simple Effects2
IV
Oral


Oral


73.2
75.1
73.8
74.4



71.8
74.5
75.8
74.3


72.0
73.3
72.4
72.9



70.7
73.2
74.0
72.5


37.5b
43.1
42.1



47.8a
47.6a


Values listed are means of 8 observations and means within the same
column and group with different superscripts are significantly
(P < .01) different.

Values listed are means of 4 observations and means within the same
column and group with different superscripts are significantly
(P < .01) different.


Manganese
Level





















49 .0
09 (N 0 0

09 4--. 4.0 -9
4*9 .-A ONI (


0 to ,n0 ,0


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rlH I -i N HC -


S C CO CO

-n -m -. 4.




C0 r'4 0N N






09 O r- 0oO
I I I


m ij in Cl










co (o on o
09 H 01 O


Hr-l o -- C4 (N 04 49D N-
ON CO N C(4 CON H ( DJ N


u
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4.4.4
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3 (3 n0i
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to r. a
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01 C (
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1' 0 H;<
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TABLE 10. MAIN AND SIMPLE EFFECTS OF DIETARY MANGANESE LEVEL AND
PATHWAY OF RADIOISOTOPE ADMINISTRATION ON
TISSUE MINERAL COMPOSITION OF SHEEP



Dietary Isotope
Dietary Isotope Tissue Minerals, ppm in Dry Matter
Mn Admin. _____ _
Level Pathway Mn Fe Cu Zn Mg Ca P


Main Effec
Low
High


ts
:ts


Liver


9.9a 1055x 42.8a 193a 588 163 11,743
44.2b 698y 62.2b 166h 608 157 12,059
IV 25.8 868 55.3 193x 606 159 11,727
Oral 28.2 885 49.7 167y 590 16] 12,050


Simple Effects2
Low IV 9.7a 1021x 49.7a 211a 588 157 11,110
Low Oral 10.0a 1090x 35.9a 175b 587 168 12,325

High IV 42.0b 715' 61.0b 175b 623 161 12,344
High Oral 46.4b 681y 63.4b 158b 592 154 11,774


Main Effec
Low
High


ts
:ts


Kidney


5.2a 268 17.4 141 739 399 11,545
22.5b 224 18.9 165 780 403 11,838
IV 13.0 260 19.3 159 762 409 11,676
Oral 14.6 232 17.1 147 757 394 11,723


Simple Effects
Low IV 4.4a 277
Low Oral 6.0a 259
High IV 21.7b 243
High Oral 23.2b 205


Main Effects
Low 2.4" 283
High 5.4b 272
IV 3.8 276
Oral 4.0 279


18.6 149 736 412 10,693
16.3 133 742 386 12,184
19.9 169 789 405 12,413
17.9 161 771 402 11,262

Heart


16.8 85 895 190 9,683
17.6 86 898 198 10,034
16.8 86 875 197 9,564
17.6 86 918 192 10,152











TABLE 10. (continued)


Dietary Isotope
Mn Admin. Tissue Minerals, ppm in Dry Matter
MLevel Panhway n Fe Cu Zn Mm Ca P
Level Pathway Mn Fe Cu Zn Mg Ca P


Simple Effects2
Low IV
Low Oral
High IV
High Oral


2.3a 271 16.6
2.5a 294 17.1
5.2b 281 17.0
5.5b 264 18.2


86 885 202
85 905 178
85 864 192
87 931 205


Spleen


Main Effects
Low 2.6a 4423
High 6.4b 3056
IV 3.6 3272
Oral 5.3 4207


Simple Effects2
Low IV
Low Oral
High IV
High Oral


2.0a 4238
3.1a 4608
5.3b 2306
7.5b 3807


9.3 141 752 225
8.2 134 717 212
8.1 133 735 223
9.4 142 734 214



8.9 139 762 241
9.6 143 742 210
7.2 127 708 206
9.3 140 727 218


Muscle


Main Effects1
Low 1.0x 130
High 1.6y 120
IV 1.2 121
Oral 1.4 129


Simple Effects2
Low IV
Low Oral
High IV
High Oral


1.1x 139
0.9x 121
1.3y 103
1.9y 138


5.9 126 802 124
4.9 120 842 127
5.4 124 847 128
5.5 122 798 123



7.0 118 839 126
4.9 133 766 121
3.8 129 855 131
6.1 112 829 124


9,439
9,927
9,690
10,376


12,701
12,972
13,341
12,378



12,787
12,614
14,079
12,142


7,345
7,971
7,652
7,663



7,348
7,342
7,958
7,983


I~ __~_










TABLE 10. (continued)


Dietary Isotope
Mn Admin. Tissue Minerals, ppm in Dry Matter
Level Pathway Mn Fe Cu Zn Mg Ca P

Bone


Main Effects1
Low

High


Simple
Low
Low
High
High


IV
Oral

Effects2
IV
Oral
IV
Oral


Main Effects1
Low
High


Simple
Low
Low
High
High


1Each
2Each
2
Each


IV
Oral

Effects2
IV
Oral
IV
Oral


5.2a
14.3b
10.1
9.4



5.0a
5.4a
15.1b
13.4b




3.1a
14.6b
8.7
9.0



2.9a
3.4a
14.6b
14.6b


2.8a
4.1b
3.1x
3.8y



2.4x
3.2y
3.8y
4.5y

Brain


14.5
1.5.5
14.5
15.4



14.0
14.9
14.9
16.0


value based on 8 observations per

value based on 4 observations per


15,484
14,820
16,299a
14,005b



16,323
14,646
16,275
13,365




643
632
647
628



652
634
642
622


312,166x
318,867y

317,659
313,374



315,107x
309,226x
320,212y
317,522y




308
305
306
307



306
309
305
304


180,796
175,396
175,617
180,575



178,072
183,521
173,161
177,630




15,721
15,280
15,431
15,423



15,952
15,491
15,170
15,390


treatment for main effects.

treatment for simple effects.


' Means within the same column and group with different superscripts
are significantly (P < .01) different.

x, Means within the same column and group with different superscripts
are significantly (P < .05) different.








manganese fed. Manganese levels were greater in the liver, kidney, heart,

spleen, hone, brain (P < .01) and muscle (P < .05) when the high

manganese diet was fed. The high manganese diet resulted in significantly

lower levels of iron (P < .05), copper and zinc (P < .01) in the liver.

However, bone levels of copper (P < .01) and calcium (P < .05) were

significantly increased by high dietary manganese levels.

Pathway of isotope administration had a significant effect on the

tissue concentration of three minerals. Those lambs given the oral dose

of 54in had significantly lower levels of zinc in the liver (P < .05)

and less magnesium in bone (P < .01), but higher levels of bone copper

(P < .05) than those given 541.In intravenously.



Discussion

The results of this study demonstrating that both 5/'4n and stable

manganese are excreted almost exclusively by way of the feces are in

agreement with previous research findings by this author (Watson, 1968a),

Cotzias (1958), and others. The increase in excretion of the radio-

isotope and stable manganese with increased dietary manganese was

expected due to mass action. With excretion primarily fecal, the finding

that the body retained an intravenously injected dose of 54Mn for a

longer period of time than an oral dose of the radioisotope was also in

accord with expectations.

The extremely rapid plasma disappearance rate of intravenously

injected 54k. is in agreement with similar studies conducted with rats

(Cotzias, 1963). The body seems to have a mechanism for homeostatic

control, maintaining low blood levels of manganese end oi-her trace

elements. Even though the plasma concentration of stable manganese was








greater for those lambs fed the higher level of dietary manganese, the

values recorded for bcth groups were very close to the "normal" range

reported by Underwood in 1962 (5 to 20 micrograms percent).

Apparent absorption and net retention values (Table 6) for stable

manganese and tissue radioisotope levels in orally dosed animals (Tables

4 and 5) indicate that absorption of manganese from the intestinal tract

is limited. This agrees with reports by Skinner, Peterson and Steenbock

(1931) using rats and by Lassiter and White (1966) using sheep. The

negative treatment averages for percent absorption and retention of

stable manganese (Table 6) are difficult to explain. However, these

findings are in agreement with previous research by this author (Watson,

1968a) and by Lassiter (1970). In this experiment both positive and

negative values for absorption and retention were recorded, but fecal

manganese was approximately equal to dietary intake of the element.

Variation was also quite high within all treatments. Skinner, Peterson

and Steenbock (1931) reported that rats excrete as much as 99 percent of

their dietary intake in the feces. Even though negative balances were

recorded, tissue deposition of both stable manganese and 54.Mn demonstrates

that manganese is absorbed from the intestinal tract. The inverse

relationship between dietary manganese level and the percent of the

radioactive manganese dose retained by various tissue is in agreement

with research with rats (Rojas, Dyer and Cassatt, 1966), chickens

(Settle et al., 1969) and swine (Plumlee et al., 1956). The relative

enrichment of tissues with radioactivity in this experiment is also in

general agreement with the results reported by these researchers. As

was noted by Cctzias (1963), those tissue which are richest in concentra-

tion of mitochondria (liver and kidney) retain more manganese. This








supports the theory that the functional activity of manganese may be

located in the mitochondria.

The decreased apparent absorption of ash or total minerals in those

lambs fed supplemental manganese is partially explained not only by the

limited absorption of manganese itself, but by the decrease in absorption

of iron and phosphorus resulting from the addition of manganese. These

results are supported by the finding of Gallup et al. (1952) with steers,

and Hartman, Matrone and Wise (1955) with lambs that excess manganese

interferes with absorption of phosphorus and iron.

The absorption values for the dietary minerals in this experiment

are similar to, but slightly lower in most cases, than values obtained

by Standish (1970) with sheep fed an experimental diet similar in

composition to the one used here. The negative absorption of iron and

zinc as well as the individual treatment differences in magnesium

absorption may be due to intakes of these elements from extraneous

sources. The animals were housed in metal metabolism cages and were

periodically observed chewing on the cages. Similar results have been

observed with lambs fed in the same cages in the past (Standish, 1970

and Watson, 1968b).

The increased concentration of manganese in the tissue of lambs fed

the higher level of dietary manganese is supported by the work of Rojas,

Dyer and Cassatt (1965) with cattle, Johnson (1943) and Svajgr, Peo and

Vipperman (1969) with swine and Mathers and Hill (1968) with poultry.

The manganese concentration in the tissue of the lambs fed the basal diet

is very similar to "normal" levels reported by the above authors as well

as Loaiza (1968) and Standish (1970).

Tissue concentrations of all the mineral elements recorded in this








experiment are similar to values reported by Loaiza (1968) with pasture-

fed cattle and by Standish (1970) with cattle fed a diet similar in

composition to the diet used in the present studies.

The decreased level of iron in the liver as a result of increased

dietary manganese agrees with results of Hartman, Matrone and Wise

(1955). Also, the lower zinc level in the livers of those lambs fed the

high manganese diet demonstrates the antagonistic effect of excess

manganese on zinc utilization reported by Pfander, Beck and Preston

(1966). The increased concentrations of copper in the liver and bone

and increased bone calcium resulting from increased dietary manganese

are in contrast to the findings of Gallup et al. (1952) and Gubler et al.

(1954). This author is unable to attach biological significance to the

effect of pathway of radioisotope administration on levels of zinc in

the liver and magnesium and copper levels in the bone since such a small

quantity of manganese was administered in the radioactive dose. No such

effects have been reported by other researchers using radioisotopes of

manganese.



Summary

Sixteen Florida Native wether lambs were employed in a 2 x 2 factor-

ially designed trial to study the metabolism and distribution of

manganese as well as some of the nutritional interrelationships which

are exhibited by the element. One hundred microcuries of 541n, a

radioisotope of miinganese, were administered either orally or intraven-

ously to lambs receiving a basal diet based on hay, corn and soybean

meal and containing 30 ppn nanganese, or the basal diet plus 4,000 ppm

supplemental mangaiese. A 21-day fecal and urinary collection period








followed dosing. A 35-day clearance period elapsed before the lambs

were again dosed with 54Mn. Blood samples were then taken at periodic

intervals for 96 hours after which all animals were killed and selected

tissues removed for analyses. Absorption of manganese from the intestinal

tract appeared to be low and excretion of both stable manganese and 54Mn

was almost exclusively by way of the feces regardless of dietary

manganese level or pathway of isotope administration. Low levels of

dietary manganese and intravenous administration of the radioisotope

resulted in the greatest tissue retention of radioactivity. The tissues,

listed in decreasing order of enrichment with radioactive manganese

were as follows: kidney, liver, spleen, brain, heart, bone and muscle.

Tissue and plasma levels of stable manganese were greater for those

lambs fed the manganese-supplemented diet than for those fed the unsupple-

mented basal diet. The high manganese diet also resulted in decreased

absorption of iron and phosphorus from the intestinal tract and decreased

liver concentrations of iron and zinc.














CHAPTER IV


EXPERIMENTS 2 AND 3. BIOLOGICAL ASSAY OF
INORGANIC MANGANESE FOR CHICKS



Many inorganic forms of manganese are available for use as supple-

ments in poultry feeds. A suitable assay for biological availability of

the element, however, is not available, and little is known of the

relative utilization of the various supplemental sources by the chick.

Early reports by Schaible, Bandemer and Dividson (1938) and Gallup and

Norris (1939a) stated that practically all the chemical forms were of

equal value to the chick except for a few carbonate and silicate ores.

In these studies, the only criterion of availability was the ability of

the manganese supplement to prevent the occurrence of perosis. More

recently, Hennig et al. (1967) reported that radioactive manganese as

54MnC12 was incorporated into the body of a chick to a significantly

greater extent than manganese as 54MnSO4 or 54MnO2. Only a few of the

products which are currently available for use as manganese supplements

were tested, and the levels of manganese added to the diets were not

sufficiently low to provide a marginal state of manganese nutrition in

which extremely sensitive responses would be expected.

Two experiments were conducted with growing chicks receiving varied

low levels of supplemental manganese from different sources. Objectives

of this research were to (1) develop a suitable biological assay for








manganese and (2) determine the relative availability of manganese from

certain inorganic sources.



Experimental Procedure

The forms of manganese tested in these experiments were reagent-

grade manganese sulfate (4MnSO4 *120), as the reference standard, and two

commercial feed-grade manganese oxides, hereafter referred to as manganese

oxide No. 1 and manganese oxide No. 2. Table 11 presents the average

manganese contents of the three sources as determined by two laboratories.

These values were used in preparing experimental diets. Other chemical

constituents, results of X-ray diffraction studies, and relative

solubilities for each of the three test materials are also shown. The

solubility of each manganese material was obtained in water, 0.4 percent

hydrochloric acid, 2 percent citric acid, and neutral ammonium citrate.

Solubility in each solvent was determined by adding 100 milliliters of

the solvent to 0.1 grams of the test material, stirring constantly for

one hour at a temperature of 37 degrees Centigrade, then filtering

through No. 42 filter paper. Manganese content was then determined on

the filtrate by atomic absorption spectrophotometry as outlined by the

manufacturer (Anonymous, 1964).

The basal diet used in both of these experiments is shown in Table

12. This semi-purified diet is similar in composition to that used by

Leach, Norris and Scott (1962) in studying the relationship of choline

to perosis in chicks. Those nutrients known to be related to the perosis

syndrome, including inositol, biotin, choline, folic acid, vitamin Bl2

and pyridroxine, were supplemented at levels well above established

requirements. The diet, as formulated, contained 4 ppm manganese.












TABLE 11. CHEMICAL COMPOSITION OF MANGANESE SUPPLEMENTS, THEIR
SOLUBILITY IN VARIOUS SOLVENTS AND X-RAY PATTERNS



Chemical Constituents, (%)

Supplement Mn Fe Ca Mg P Cu Zn



MnSO .H 0 31.16 0.03 0.002 0.001 --- 0.052 0.004
MnO No. 1 51.95 5.63 0.042 0.123 0.402 0.112 0.060
MnO No. 2 36.10 5.46 0.816 0.441 0.223 0.065 0.098




Solubility, (%)

0.4% 2% Neutral
Water Hydrochloric Citric ammonium Interpretation
acid acid citrate of X-ray Patterns


100.0
1 0.0


100.0
35.4


0.0 52.0


100.0 100.0 MnSO4.H20
77.5 29.1 MnO (Manganosite)
and Mn3 0
(Hausmannit e)

87.4 16.5 MnO, Mn304,
and Quartz


MnSO .H20
MnO No.


MnO No. 2















TABLE 12. COMPOSITION OF BASAL DIET1


Ingredient %


Glucose

Soybean protein2

Corn oil

Cellulose

Vitamin mixture3

Mineral mixture4

Glycine

DL Methionine


Test diets contained the experimental manganese source
expense of glucose.


added at the


Assay Protein, C-l, Skidmore Enterprises, Cincinnati, Ohio.

Supplied the following per kg of diet: inositol, 1510 mg.; niacin,
99.2 mg.; thiamine HCI, 22.1 mg.; riboflavin, 22.1 mg.; Ca pantothenate,
66.2 mg.; pyridoxine HCI, 22.1 mg.; menadione sodium bisulfite, 49.6 mg.;
vitamin A, 19,846 I.U.; vitamin D3, 2205 I.U.; alpha tocopherol, 110.3
mg.; ascorbic acid, 992.3 mg.; choline chloride, 1653 mg.; p aminoben-
zoic acid, 110.3 mg.; biotin, 0.44 mg.; folic acid, 1.98 mg.; and vitamin
B12, 0.03 mg.

Supplied the following per kg of diet: CaHP04, 1.70 gm.; CaC03, 1.83 gm.;
KH2P04, 1.38 gn.; NaCI, 0.60 gm.; MgS04, 0.25 gm.; FeS04-7H20, 33.30 mg.;
KI, 0.26 mg.; CuS04'51120, 1.67 mg.; CoC12"6H20, 0.17 mg.; Na2Mo04 21120,
0.83 mg.; and ZnO, 7.47 mg.


57.97

27.00

3.00

3.00

2.20

5.83

0.30

0.70

100.00








Experimental treatments were prepared by adding graded levels of the

three manganese sources to the basal diet at the expense of glucose.

Each experiment consisted of eight treatments and each treatment

included three replicates of ten chicks which were randomly assigned to

experimental groups. The eight treatments tested were: basal diet,

basal plus 10, 20 and 30 ppm of manganese supplied from manganese sulfate,

basal plus 10 and 20 ppm from manganese oxide No. 1, and basal plus 10

and 20 ppm from manganese oxide No. 2. The levels of manganese in all

diets were verified by chemical analysis.

Two-hundred-forty (240) day-old Leghorn cockerel chicks were

obtained from a local commercial hatchery for each experiment. Treat-

ment replicates were randomly assigned to pens in a thermostatically

controlled, electrically heated battery brooder constructed primarily of

stainless steel with raised wire floors. All chicks received their

respective experimental diet and tap water (0.4 ppm manganese) ad libitum

throughout the 28-day trial. After 28 days, body weights were recorded

and legs of all chicks were individually examined for abnormal develop-

ment or perosis. Upon visual examination, each chick was assigned a

value within the range of from zero to four, depending upon the degree

of abnormal leg development. A value of zero was assigned when the leg

appeared completely normal. A value of one was given when there was a

slight amount of swelling of the tibiometatarsal joint, two was assigned

when there was a marked degree of swelling of the joint, three when there

was swelling plus a slight amount of slipping of the achilles tendon,

and four when swelling was combined with a marked degree of slipping of

the tendon.

Chicks were sacrificed after the examination for perosis. In









Experiment 2, the right tibiae were removed from seven chicks on each

treatment for determination of percent bone ash. Bone ash and bone

manganese levels were determined on the combined right tibiae and femur

of all 30 chicks on each treatment in Experiment 3. Breaking strength

was also determined on the left tibiae of all chicks in Experiment 3 as

described by Rowland et al. (1967). Bones were ashed according to the

method described by A.O.A.C. (1960). Bone manganese content was deter-

mined by atomic absorption spectrophotometry.

The data for body weights, bone abnormalities, bone ash, breaking

strength, and bone manganese were subjected to analysis of variance, and

bone manganese data were subjected to regression and correlation analyses

as outlined by Steele and Torrie (1960). Significant differences between

treatment means were determined by Duncan's multiple range test (1955).



Results

Statistical analyses of the data obtained from body weight and leg

abnormality measurements revealed no experiment x treatment interactions

so the data from the two experiments were combined for presentation.

The results of the body weight determination at 28 days of age are shown

in Table 13. Average weight of the chicks receiving the basal diet (252

grams) was lower (P < .01) than that of the other treatment groups.

Chicks fed the basal diet with 10 ppm manganese from manganese oxide No.

2 had an average weight of 288 grams which was lower (P < .01) than the

weight of the group receiving the basal plus 20 ppm manganese from the

same source. There were no significant differences in average weights

among treatment groups fed any other supplemental level or source of the

element.














TABLE 13. BODY WEIGHTS OF 28-DAY-OLD CHICKS FED VARIOUS FORMS AND
LEVELS OF SUPPLEMENTAL LMAGANESE
(EXPERIMENTS 2 AND 3)1




Manganese Supplemental Manganese (ppm)
Source
Source 10 20 30


Body wt., gm

MnS04.H20 252a 306b,c 308b,c 317h,c

MnO No. 1 --- 302b,c 316bc --

MnO No. 2 -- 288b 323c




Values listed represent an average of 60 chicks and means bearing
different superscripts are significantly different (P < .01).









The incidence of leg abnormalities was related to the response

obtained for growth rate (Table 14). The value for degree of leg

abnormalities in the groups fed the basal diet was higher (P < .01) than

for any other treatment group. Also, those chicks receiving the basal

plus 10 ppm manganese from the manganese oxide No. 2 had a higher (P <

.01) assigned value than the chicks fed any other level or source of

supplemental manganese. There were no significant differences among any

of the other treatment groups in the incidence of leg abnormalities.

Statistical analyses of the data obtained from bone ash determina-

tions revealed a significant experiment x treatment interaction; thus

the data from the two experiments arc presented separately (Table 15).

Dietary levels and source of manganese had no effect on percent bone ash

of chicks in Experiment 2 when the comparison was based on the data of

seven chicks per treatment. In Experiment 3, xith 30 observations per

treatment, bone ash response was similar to that obtained for growth

rate and leg development. The treatment groups fed the basal diet and

those fed the basal plus 10 ppm of the element from manganese oxide No.

2 had the lowest levels of bone asl and were not significantly different.

The highest level of bone ash was found in those chicks receiving 20 ppm

of the element from manganese oxide No. 2, and all other treatment groups

were intermediate in bone ash response.

Treatment averages for tibiae breaking-strength determinations in

Experiment 3 are presented in Table 16. The average breaking strength of

the tibiae for all treatments was 10.5 pounds, and statistical analyses

of these values revealed no significant differences among treatments.

Table 17 presents the average values for bone manganese for all

treatments in Experiment 3. Treatment differences were more pronounced













TABLE 14. LEG ABNORMALITY SCORES OF 28-DAY-OLD CHICKS FED VARIOUS FORMS
AND LEVELS OF SUPPLEMENTAL MANGANESE (EXPERIMENTS 2 AND 3)1,2


Manganese
Source


Supplemental Manganese (ppm)
10 20


Leg Score, (0-4)

MnSO H20 3.01a 0.75c 0.55 0.27c

MnO No. 1 --- 0.76c 0.66c

MnO No. 2 --- 1.53b 0.57c


Grading Scale:


Normal leg
Slight swelling of tibiometatarsal joint
Marked swelling of joint
Swelling and slipping of tendon
Swelling and marked slipping of tendon


Values listed represent an average of 120 observations and means
bearing different superscripts are significantly different (P < .01).














TABLE 15. BONE ASH OF 28-DAY-OLD CHICKS FED VARIOUS FORMS AND LEVELS
OF SUPPLEMENTAL MANGANESE




Manganese Supplemental Manganese (ppm)
Source
source 10 20 30


Experiment 21


% in dry, fat-free tibia

47.6 48.3 46.7 46.3

--- 46.9 46.6 -

--- 47.0 46.4



Experiment 32


% in dry, fat-free tibia and femur


45.8a,b


ecd
47.4c'd

46.2bc

44.7a


46.7b,c,d


Values listed are treatment averages based on 7 observations per
treatment.

Values listed are treatment averages based on 30 observations per
treatment. Means with different superscripts are significantly
different (P < .01).


MnSO H20

MnO No. 1

MnO No. 2


MnSO4 H20

MnO No. 1

MnO No. 2


47.5 d

47 .2"

47.7d
















TABLE 16. TIBIA BREAKING STRENGTH OF 28-DAY-OLD CHICKS FED VARIOUS
FORMS AND LEVELS OF SUPPLEMENTAL MANGANESE1




Manganese Supplemental Manganese (ppm)
Source 0 10 20 30


(pounds)

MnSO4.H20 11.5 11.8 9.4 9.4

MnO No. 1 --- 10.4 8.3 --

MnO No. 2 --- 11.2 11.5 ---




1Values represent means of 30 observations per treatment.













TABLE 17. BONE (TIBIA PLUS FEMUR) MANGANESE LEVELS OF 28-DAY-OLD CHICKS
FED VARIOUS FORMS AND LEVELS OF SUPPLEMENTAL MANGANESE1




Manganese Supplemental Manganese (ppm)
Source 0 10 20 30


(ppm in dry, fat-free bone)

MnSO .H20 2.46a 2.88b'c 3.30d 3.81e

MnO No. 1 --- 2.95b,c 3.17d,c

MnO No. 2 --- 2.74b 3.39d



(ppm in bone ash)

MnSO .H20 5.11a 6.08a,b 6.80a,b, 8.17e

MnO No. 1 --- 6.27a,b 6.71a,b,c --

MnO No. 2 -- 6.12a'b 7.10a'b'c



Values listed are treatment averages of 30 observations and means
bearing different superscripts are significantly different (P < .05).









when bone manganese was expressed as ppm in dry, fat-free bone rather

than ppm of manganese in bone ash. The manganese level in the dry, fat--

free bone of chicks fed the basal diet with no supplemental manganese

(2.46 ppm) was lower (P < .05) than the level found in any other treat-

ment group. For those groups receiving supplemental manganese from the

standard manganese sulfate or from manganese oxide No. 2 the manganese

level in dry, fat-free bone directly reflected the changes in dietary

manganese levels (P < .05). The same numerical trend was demonstrated

for those groups receiving manganese oxide No. 1. When expressed as

ppm in bone ash, bone manganese levels also increased numerically in

every case with increasing dietary levels of the element. The manganese

level in bone ash of chicks fed only the basal diet was significantly

lower (P < .05) than that of those receiving 30 ppm of the element from

the standard manganese sulfate. Those fed 30 ppm supplemental manganese

from manganese sulfate also had a higher level (P < .05) of the element

in the bone ash than any group receiving 10 ppm of added manganese from

any source.

The relationship between dietary levels of manganese as manganese

sulfate and bone manganese is shown graphically in Figure 4. Significant

(P < .01) correlations of 0.665 and 0.667 existed between manganese

intake and level of bone manganese when expressed as ppm in the dry, fat-

free bone or ppm in bone ash respectively. The linear regressions

obtained are shown as follows: Y = 2.28 + 0.044X and Y' = 4.82 + 0.091X

where X is equal to the dietary manganese level, Y equals the level of

manganese in dry, fat-free bone, and Y' the level of manganese in bone

ash.


















8.0






p6.0


p.









2.0
ai




2.0


Dietary Manganese, ppm


Figure 4. Relation of Bone (tibia plus femur) Manganese Levels to
Manganese Intake in the Form of Manganese Sulfate.









Discussion

These data are in agreement with those published recently by Settle

et al. (1969) which showed that a similar semi-purified diet, when fed

alone, was totally inadequate in supporting maximum growth rate and in

preventing the occurrence of perosis. From the data obtained in the

studies reported herein, it is concluded that 10 ppm manganese added to

the diet used in these experiments from either the sulfate or the

manganese oxide No. 1 source ~ere adequate to promote normal growth rate

and leg development. These two sources of manganese were more available

to the chick than was the manganese oxide No. 2 since 10 ppm manganese

from the latter source produced a lower growth rate and a higher

incidence of perosis. Results of bone ash determinations are in general

agreement with the results of measurements of growth rate and incidence

of perosis; however, results were not as well defined. Percent bone ash

was generally increased by the addition of manganese to the basal diet

from each source except at the level of 10 ppm from manganese oxide

No. 2.

Bone manganese levels were more directly related to dietary levels

of the element than were growth rate or leg development since a signi-

ficant linear increase in bone manganese resulted from increasing

dietary levels of supplemental manganese as reagent-grade manganese

sulfate from zero to 30 ppm (Figure 4). A significant linear response

was not found with body weights (Table 13), leg abnormality scores

(Table 14) or bone ash (Table 15). This indicates that levels of

manganese in the bone may be a sufficiently sensitive response criterion

to detect small differences in dietary levels or differences in biological

availability of manganese. These results suggest that a biological assay









for manganese may be developed using manganese sulfate as a reference

standard and bone manganese levels along with growth rate and leg

development as the response criteria.

Growth rate and leg development were directly related to the solu-

bilities of the test material only in the case of neutral arrmonium

citrate. Manganese oxide No. 2 added at a level of 10 ppm manganese

was less available than manganese oxide No. 1 as measured by growth rate

and leg development. However, manganese oxide No. 2 was more soluble in

0.4 percent hydrochloric acid and 2 percent citric acid than was manganese

oxide No. 1. Thus, solubility of manganese sources in these solvents

appears to be of limited usefulness in predicting availability of the

element.



Summary

Two experiments were conducted in an effort to develop a suitable

biological assay for manganese availability with chicks. Reagent-grade

manganese sulfate was used as the reference standard and was added to

a semi-purified basal diet at levels of 10, 20 and 30 ppm manganese.

Two commercial feed-grade manganese oxides were the other test materials,

and were added at the levels of 10 and 20 ppm manganese to the same basal

diet. Ten ppm supplemental manganese were adequate for normal growth and

bone ash and to prevent the occurrence of perosis when supplied from the

sulfate or one of the oxide sources. Ten ppm supplemental manganese from

the other oxide source, however, resulted in reduced growth rates, a

lower percent bone ash and a higher incidence of perosis.

Manganese concentration in the bone increased with increasing dietary

levels of the element. This response suggests that the level of manganese




66



in the bone may be a sufficiently sensitive response criterion to be

used in a manganese assay using manganese sulfate as the standard source.















CHAPTER V


EXPERIMENTS 4 AND 5. BIOLOGICAL AVAILABILITY OF SEVERAL
INORGANIC FORMS OF MANGANESE TO CHICKS



Two experiments were conducted to determine the availability of

manganese from several inorganic sources using the assay method suggested

from the results of Experiments 2 and 3. An attempt was also made to

demonstrate the relationship between the response criteria and levels of

manganese intake above those tested in the first two experiments.



Experimental Procedure

The basal diet used in the two identical experiments was the same

as that used in Experiments 2 and 3 (Table 12). Upon analysis this diet

was found to contain 5 ppm manganese. Treatment diets were prepared by

adding the supplemental manganese source at the expense of glucose. The

sources of manganese tested are shown in Table 18 along with the manganese

content of each source, relative solubility in various solvents, and

results of X-ray diffraction studies. Manganese content and solubility

were determined as outlined in the experimental procedure for Experi-

ments 2 and 3.

Each experiment consisted of 12 treatments and each treatment

included two replicates of 10 chicks which were randomly assigned to

experimental groups. The 12 treatments tested were basal diet, basal














44
d
44

441
441
Pu





X
41
0
0






*rl
44
a
'4-)





a,
0

44
0
H
(U

4J
C4
44


c.4 m
0 0
*, U


N
4J u4
X rx






(h a)
U




0 c.


00
ni










0 o


0 D








v 0 o


-f co


44

U-r





4I ..





c A
f0 0
44


m 0





(' ,-
P^ C






0 C4
*4 g U
ca 0^&


U
0






Q) Q
0 -t



O 4
0 ".









H 3


CO C00


cO










o Coo


rC o


o o o o o


0 0


"0
a


04 m








C 4C 0r


-:t
0
H 4


LO a



U 44



0 H



0 3


6CO 0


a0 0


H4e

: 0 4J

zau







NU









I-r-
0

-cj c
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o
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a4

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plus 10, 20, 30, 60 and 120 ppm manganese supplied from the reference

standard, reagent-grade manganese sulfate, and basal plus 10 ppm supple-

mental manganese supplied from six other sources referred to as sources

No. 2, 4, 6, 8, 12 and 13 as listed in Table 18. The manganese level of

each diet was verified by chemical analysis.

As in Experiments 2 and 3, 240 day-old Leghorn cockerel chicks

obtained from a local commercial hatchery were used in each experiment.

Treatment groups were housed in a thermostatically controlled, electri-

cally heated battery brooder constructed primarily of stainless steel

with raised wire floors. Tap water (0.4 ppm manganese) and the experi-

mental diets were supplied ad libitum to all chicks throughout the 28-

day experimental period. At the end of the 28-day trial, individual body

weights were recorded and all chicks were examined for abnormal leg

development or perosis and scored according to the method outlined for

Experiments 2 and 3. All chicks were killed after examination for perosis

and the right tibiae and femurs removed for determination of bone ash

and bone manganese levels as described for Experiments 2 and 3.

The data resulting from measurements of body weight, leg abnormal-

ities, bone ash and bone manganese were subjected to analysis of variance

and bone manganese data analyzed by regression and correlation analyses

as outlined by Steel and Torrie (1960). Significant treatment differences

were determined by Duncan's multiple range test (1955).



Results

Statistical analyses of the data obtained from these experiments

revealed a significant (P < .01) experiment x treatment interaction,

therefore the data are presented separately.








The results of body weight determinations at 28 days are shown in

Table 19 for both experiments. Those chicks receiving only the basal

diet in Experiment 4 had an average body weight of 195 grams which was

significantly (P < .01) lower than any other treatment group. There were

no significant differences in body weights of 28-day-old chicks fed 10

ppm supplemental manganese either from the standard source or from

sources No. 2, 4, 6 and 12 or 20 ppm from the standard source. There

were no significant differences among groups fed 10 ppm manganese from

sources No. 8 and No. 13 or 30 and 120 ppm from the standard source.

Body weights were significantly (P < .01) greater for the chicks receiv-

ing 60 ppm supplemental manganese from the standard source (277 grams)

than for those receiving the basal or basal plus 10 ppm manganese from

the standard source or No.'s 2, 4, 6 and 12. Body weights were generally

higher for 28-day-old chicks in Experiment 5. As in Experiment 4, feed-

ing the basal with no supplemental manganese resulted in the lowest

(P < .01) average value for 28-day body weight (227 grams). In contrast

to the results of Experiment 4 however, there were no significant

differences in body weights among the other treatment groups with an

average weight of 271 grams recorded for these groups.

A summary of leg abnormality scores for both experiments is presented

in Table 20. The group fed the basal diet in Experiment 4 had the highest

incidence of leg abnormalities (2.78) but was not significantly different

from groups receiving 10 ppm manganese from sources No. 2, 4, 6 and 12.

There were no significant differences among the groups fed 10 ppm supple-

mental manganese from any source with an average value of 1.99 recorded

for all of these treatments. Leg abnormalities were, for all practical

purposes, absent in chicks fed more than 10 ppu supplemental manganese

front the standard source in Experiment 4.














TABLE 19. BODY WEIGHTS OF 28-DAY-OLD CHICKS AS INFLUEN ED Ef
SOURCE OR LEVEL OF SUPPLEMENTAL MANGANESE


Supplemental Manganese, ppm
0 10 20 30 60 120


Experiment 4
(Body Wt., gm)
195a 241b,c 256b,c,d 265c,d 277d 271cd
--- 240b,c --
--- 228b
226b ---
--- 270 c,d

--_- 240bc -- ---
--- 265 c,d

Experiment 5


Manganese
Source





Standard
No. 2
No. 4
nb. 6
No. 8
Nlo.12
No.13




Standard
No. 2
No. 4
No. 6
No. 8
No.12
No.13


(Body Wt., gm)
289b 265b


Values listed are means of 20 observations and different superscripts
within experiment groups denote significant (P < .01) differences.


280b
259b
271b
268b
283b
259b
266b













TABLE 20. LEG ABNORMALITY SCORES OF 28-DAY-OLD CHICKS AS
INFLUENCED BY SOURCE OR LEVEL OF SUPPLEMENTAL MANGANESE1,2


Supplemental Manganese, ppm
0 10 20 30 60 ]20

Experiment 4
(Leg Score, 0-4)
2.78a 1.58b 0.23C 0.32c 0.28C 0.18c
--- 2.48a,b
--- 1.92a,b
2.25a,b

1.60 -

--_- 2.30a,b ....
--- 1.78b 5


Experiment 5


Manganese
Source




Standard
No. 2
No. 4
No. 6
No. 8

No. 12
No. 13





Standard

No. 2
No. 4


(Leg Score, 0-
0.08f


-4)
0.01 0.09


1.01c,d,e

0.43d,e,f

2.03b
1.18cd


Values
within


listed are means of 20 observations and different superscripts
experiment groups denote significant (P < .01) differences.


Grading Scale: 0 = Normal leg
1 = Slight swelling of tibiometatarsal joint
2 = Marked swelling of joint
3 = Swelling and slipping of tendon
4 = Swelling and marked slipping of tendon


3.20a 0.35ef
--- 3.12a
1.64bc


------- -


0.22f









Feeding the basal diet or basal plus 10 ppm manganese from source

No. 2 resulted in the highest (P < .01) incidence of leg abnormalities

in Experiment 5. An average value of 2.03 was assigned to those chicks

fed 10 ppm manganese from source No. 12. This was significantly (P < .01)

greater than values assigned to those fed 10 ppm from the standard or

from sources No. 6, 8 and 13 (0.74). However, this was not significantly

different from the leg score for the group fed 10 ppm manganese from

source No. 4. Again there was practically no occurrence of perosis in

the groups fed more than 10 ppm supplemental manganese, and in this

experiment these groups were not significantly different from those fed

10 ppm from the standard source or from source No. 8.

Figure 5 shows representative tibiae from the chicks receiving the

basal diet and basal plus 10, 20, 30, 60 and 120 ppm manganese supplied

from the standard source, manganese sulfate, in Experiment 5. The

basal group had nuch shorter, thicker tibiae that were more bent at the

ends than any of the groups fed supplemental manganese. The tibia

length seemed to increase slightly with increasing levels of dietary

manganese up to 20 ppm even though there were no differences in the

assigned values for degree of leg abnormalities (Table 20) among the

groups receiving varying levels of supplemental dietary manganese.

Representative tibiae from the groups fed 10 ppm manganese from the

various test sources in Experiment 5 are shown in Figure 6. As was

demonstrated by visual examination of the live bird (Table 20), 10 ppm

manganese from sources No. 2, 4 and 12 resulted in shorter more extremely

bent tibiae than did 10 ppm supplied from sources No. 8 and 13. However,

the shorter tibiae for those chicks fed source No. 6 does not agree with

the lower leg score assigned to live birds on this treatment.





















































Figure 5. Representative Tibiae from 28-Day-Old Chicks Raised on
Varying Levels of Dietary Manganese in Experiment 5.




















































Figure 6. Representative Tibiae from 28-Day-Old Chicks Raised on a Diet
Containing 10 ppm Supplemental Manganese Supplied from Various
Sources in Experiment 5. Manganese Sources Represented from
Left to Right are: No.'s 2, 4, 6, 8, 12 and 13.









Statistical analyses of the data obtained from bone ash determina-

tions in Experiment 4 (Table 21) revealed no significant differences in

percent bone ash among those groups fed the basal diet, any level of the

standard source, or 10 ppm manganese from sources No. 8 and 12. These

groups had an average of 46.9 percent bone ash. Those fed 10 ppm from

source No. 6 (45.6 percent) had significantly (P < .01) less bone ash

than the group fed source No. 4 (48.1 percent). Groups receiving sources

No. 2 and 13 were intermediate in response.

Chicks fed the basal diet or basal plus 10 ppm supplemental

manganese from the standard source or sources No. 2, 4, 8, 12 and 13 in

Experiment 5 did not differ significantly in bone ash content (Table 21),

averaging 46.2 percent. These groups were significantly lower in per-

centage of bone ash than the group fed 20 ppm manganese from the standard

source (47.7 percent). Those receiving 10 ppm from source No. 6 or 30,

60 or 120 ppm of the element from the standard source had intermediate

responses in bone ash levels.

Bone manganese data, expressed as ppm in dry, fat-free bone for

these two experiments, are summarized in Table 22. Those chicks in Experi-

ment 4 fed 10 ppm supplemental manganese supplied from source No. 2 had

the lowest level of bone manganese. This level of 2.76 ppm was signifi-

cantly (P < .01) less than the bone manganese concentrations of 3.56 and

3.47 ppm found for those receiving 10 ppm supplemental manganese from

the standard source or source No. 13, respectively. There were no

significant differences among the other treatment groups fed the basal

diet or any level of the standard source less than 60 ppm. At 60 and

120 ppm however, bone manganese concentrations increased significantly

(P < .01) with increases in dietary manganese.











TABLE 21. PERCENTAGE BONE ASH IN TIBIA AND FEMUR FROM 28-DAY-OLD
CHICKS AS INFLUENCED BY SOURCE ?R LEVEL OF
SUPPLEMENTAL MANGANESE


Supplemental Manganese, ppm
0 10 20 30 60 120


Experiment 4
(Percent in dry, fat-free bone)

46.8a,b,c 46.7a,b,c 46.9a,b,c 46.7a,b,c 47.3a,b,c 47.2a,b,c
--- 46 .1a,b,c

,8.1c ... .
45.6a ---
46.4a,b,c
--- 46.9a,b,c ....... ....
-- 47.6b,c





Experiment 5


Manganese
Source






Standard
No. 2

No. 4
No. 6

No. 8
No. 12

No. 13







Standard
No. 2
No. 4

No. 6

No. 8
No. 12
No. 13


(Percent in dry, fat-free

46.3a 47.7b 47.1a,b
45.9a

45.9a
47.0a,b -
46.4a

46.3a
46.3a


bone)

47.0a,b


47.a,b


46.0a


Values listed are means of 20 observations and different superscripts
within experiment groups denote significant (P < .01) differences.


______




78







TABLE 22. BONE (TIBIA PLUS FEMUR) MANGANESE LEVELS OF 28-DAY-OLD
CHICKS AS INFLUENCED BY SOURCE OR LEVEL OF
SUPPLEMENTAL MANGANESE1


Supplemental Manganese, ppm
0 10 20 30 60 120

Experiment 4

(ppm in dry, fat-free bone)
3.27a,b,c 3.56c 3.53c 3.84c 5.24d 6.02e
--- 2.76a
--- 2.83a,b ..


--- 3.43a,b,c
.... 3.14a,b,c


3.47bc -




Experiment 5


Manganese
Source





Standard
No. 2
No. 4
No. 6
No. 8
No. 12
No. 13







Standard

No. 2
No. 4
No. 6
No. 8
No. 12
No. 13


in dry, fat-free
4.31c 5.05d


bone)
5.82e


Values listed are
within experiment


means of 20 observations and different superscripts
groups denote significant (P < .01) differences.


(ppm
3.49b
2.80a
3.03a,b
3.34a,b
3.06ab
2.96a,b
3.26a,b









The concentration of manganese in dry, fat-free bone in Experiment

5 (Table 22) was, as in Experiment 4, significantly (P < .01) lower for

the group fed 10 ppm supplemental manganese from source No. 2 (2.80 ppm)

than in those chicks fed 10 ppm from the standard source (3.49 ppm).

Groups fed the basal diet or basal plus 10 ppm added manganese from any

of the other sources were intermediate in bone manganese levels and were

not significantly different. As the level of supplemental manganese

supplied from the standard source increased from 10 to 120 ppm, there

was a corresponding significant (P < .01) rise in manganese levels of

dry, fat-free bone.

Table 23 presents bone manganese levels for Experiments 4 and 5

expressed as ppm in bone ash, The level of manganese in bone ash for

those chicks in Experiment 4 receiving 10 ppm manganese from source No.

4 was 5.91 ppm. This level was significantly lower than the" level of

7.56 ppm recorded for those receiving 10 ppm from the standard source.

There were no significant differences in manganese concentration in bone

ash for the chicks fed the basal diet or basal plus 10 ppm of the element

from any other source, with an average level of 6.98 ppm for these groups.

Significant differences did not exist among the groups fed less than 60

ppm added manganese from the reference source. However, manganese levels

in bone ash increased significantly (P < .01) to 11.10 and 12.90 ppm as

supplemental levels increased to 60 and 120 ppm respectively.

Treatment responses for manganese concentration in bone ash in

Experiment 5 (Table 23) were identical to the responses obtained when

concentration was based on dry, fat-free bone (Table 22). The level of

manganese was 6.10 ppm in bone ash for those chicks fed 10 ppm supple-

mental manganese from source No. 2. This was significantly (P < .01)









lower than the level of 7.56 ppm found for those fed 10 ppm from the

standard source. Again those fed the basal diet or 10 ppm added manganese

from the other sources were not significantly different and had an

average bone ash manganese concentration of 6.67 ppm. Bone manganese

levels rose significantly (P < .01) from 7.56 ppm for those fed 10 ppm

of the element from the standard source to 9.14, 10.75, 12.39 and 14.79

ppm as the supplemental manganese level increased from 10 to 20, 30, 60

and 120 ppm, respectively.

Significant (P < .01) correlations of 0.744 and 0.684 were found

between the level of dietary manganese supplied from manganese sulfate

and level of bone manganese (Figure 7) in Experiment 4. These correla-

tion coefficients are for bone manganese expressed as ppm in dry, fat-

free bone and ppm in bone ash respectively. In Experiment 4 the relation-

ship between bone manganese levels and dietary manganese intake was

found to be linear in nature. The linear regression equations obtained

are as follows: Y = 3.21 + 0.023X and Y' = 6.93 + 0.048X where X

represents the dietary manganese level, Y is the manganese level in dry,

fat-free bone and Y' is the level of manganese in bone ash.

The relationships between dietary manganese intake and bone manganese

levels in Experiment 5 are shown graphically in Figure 8. The "best-

fitting" regression lines were found to be quadratic over the entire

range of manganese intakes tested in this experiment (5-125 ppm).

Statistical analyses of these data revealed significant (P < .01)

correlations of 0.836 and 0.822 between manganese intake and manganese

levels in dry, fat-free bone and bone ash respectively. Quadratic

regression equations, Y = 4.73 1- 0.074 (X-44.47) 0.0003 (X-44.47)2 and

Y' = 10.3] + 0.151 (X-44.47) 0.0006 (X-44.47)2, were obtained where X




80







TABLE 23. BONE (TIBIA PLUS FEMUR) MANGANESE LEVEL OF 28-DAY-OLD
CHICKS AS INFLUENCED BY SOURCE OR LEVEL OF
SUPPLEMENTAL MANGANESE


Supplemental Manganese, ppm
0 10 20 30 60 120


Experiment 4


7.03a,b,c


7.56b,
6.00a,b
5.91a
7.46a,b,c
7.40a,b,c
6.69a,b,c
7.32a,b,c


Manganese
Source





Standard
No. 2
No. 4
No. 6
No. 8
No. 12
No. 13


11.10d


7.56b
6.10a
6.58a,b
7.10a,b
6.59a,b
6.39a,b
7.05a,b


1
Values listed are means of 20 observations and
within experiment groups denote significant (P


different superscripts
< .01) differences.


(ppm in bone ash)

7.53b,c 8.23c













Experiment 5
(ppm in bone ash)
9.14c 10.75d


Standard
No. 2
No. 4
No. 6
No. 8
No. 12
No. 33


6.32ab


12.39 e






























caa
0


a)) Q)
V) Ell


cja 41



co
0 q- Cl

*- c14 lo CD ;jC
c)













0 0,
'-q 0








0 '
5r C)C)- C) '-)












CCD





.4 r-.C)
*~ I CC)


C .4 ) 44r




















co l T C4 D o
-- H,


















1- 1 -4 C )
wdd + C)



I U
~ rl ~ o ,- Hd
WI C) .C) i l '4
C)-l
Cr
0 4 E 1-4o
C)' C)

tC C)
a'4-44-4




C L'
C) rjf\ Bl


I a r
Co



C')~~ H
md ~ 'CS)CfW~oo























o


\ D u-
*oo

\. II II

,,





















oo $
















-,- r c--




tdd '0)s0u)CuC2 auog


0)






CO



















xM
c
omo
[I I M














Cl CZ
l 00 -









o



mc
ci


Lfl1 s-l 0
t0 P










\1









is the manganese intake, Y is the concentration of manganese in dry, fat-

free bone and Y' is the ppm of the element in bone ash.

Linear regression lines were the "best-fit" in Experiment 5 when

bone manganese responses due to supplemental manganese at levels of 30

ppm and below were examined alone. The regression equations were Y =

2.48 + 0.072X and Y' = 5.47 + 0.149X where X is the manganese intake

level, Y is the manganese level in dry, fat-free bone, and Y' is the

level in bone ash.



Discussion

Identical results in chick response were not obtained in Experiments

4 and 5. Variation within treatments was quite high in Experiment 4.

Chicks in this experiment started growing at slow rate with much variation

in growth within each pen of chicks. It was suspected that these chicks

were "chilled" during transport from the hatchery to the experimental

facilities.

The results of these experiments are in general agreement with the

data obtained from Experiments 2 and 3. The basal diet, without supple-

mental manganese, resulted in less than maximum growth rate and was

ineffective in preventing the occurrence of perosis. Growth rate was

improved significantly by the addition of 10 ppm supplemental manganese

from any of the test sources. However, supplemental manganese levels

greater than 10 ppm from the standard source failed to improve growth

rate further. Results of visual examination of chicks for perosis at

28-days of age and examination of tibiae removed from these chicks

indicated that 20 ppm or more supplemental manganese prevented perosis.

The test manganese sources differed in their effectiveness in preventing









the disorder when supplemented at the 10 ppm level indicating differences

in biological availability. It appears that the standard source, source

No. 8 and No. 13 were more available or at least were more effective than

the other test sources in reducing the occurrence of perosis. Source

No. 2 was the least effective in decreasing the number of leg abnormali-

ties.

Conclusions concerning availability of manganese from the different

sources cannot be drawn on the basis of bone ash response. In general,

there were no differences in percent bone ash due to level or source of

manganese.

Bone manganese levels reflected dietary levels of the element more

directly than did growth rate or degree of leg abnormalities. This was

especially true in Experiment 5 where responses were the same if bone

manganese were expressed on the basis of either dry, fat-free bone or

bone ash. Increases in dietary manganese in Experiment 5 resulted in

significant increases in bone levels of the element in those chicks fed

the standard source as was found in the previous experiments. As indi-

cated by examination for perosis, source No. 2 was less available than

the other test sources. This source generally resulted in bone manganese

levels lower than those found when the standard source was fed at the

same dietary level and not different from levels in the basal group.

The regression and correlation analyses for these experiments are also

in agreement with the findings from the previous experiments. In Experiment

4 there was not a significant linear relationship between bone manganese

and manganese intake at dietary levels below 35 ppm. However, in Experiment

5 as in the earlier work, treatment means fell on the linear regression

line in almost every case when the effects of dietary manganese intake,










TABLE 24. RELATIVE AVAILABILITY OF MANGANESE TEST SOURCES AT A
SUPPLEMENTAL LEVEL OF 10 PPM1






Response Criteria

Mn Level in Mn Level in
Dry, Fat-free Bone Bone Ash
Manganese
Source Supplemental Supplemental Supplemental Supplemental
Plus Basal Minus Basal Plus Basal Minus Basal

Experiment 4


Standard2
No. 2
No. 4
No. 6
No. 8
No. 12
No. 13




Standard2
No. 2
No. 4
No. 6
No. 8
No. 12
No. 13


100.00 100.00 100.00 100.00
77.48 0 78.42 0
79.45 0 77.24 0
95.73 34.49 97.50 60.25
96.29 43.12 96.72 47.75
88.15 0 87.44 0
97.41 60.37 95.67 31.08


Experiment 5


100.00
78.34
84.78
93.45
85.62
82.82
91.21


100.00
0
25.18
67.82
29.31
15.55-
56.82


100.00
79.24
85.47
92.23
85.60
83.00
91.58


100.00
0
24.70
59.72
25.38
11.91
56.35


1
Availability values of zero indicate that response was less than that
of the basal group.
2
Values shown for test sources are percentages relative to the standard
response.









at 35 ppm and below, on bone manganese were examined. The quadratic

relationship that was obtained as intakes of manganese increased in

Experiment 5 was expected since this type of relationship is found with

most biological systems.

These results indicated, as was suggested in Experiments 2 and 3,

that bone manganese levels can be used as a criterion in a biological

assay testing manganese availability. It is also concluded that tests

for differences in availability should be conducted with levels of

dietary manganese below 35 ppm since it is in this range that maximum

response is obtained.

The relative availability of manganese from the various sources is

shown in Table 24. The standard values, assumed to be 100, are those

obtained from the regression analyses. Relative availability percentages

were assigned at the 10 ppm level of supplemental manganese either

including or excluding the effect of the basal diet which contained

approximately 5 ppm manganese. In Experiment 5 the relationship between

responses to sources was the same whether measured on the basis of

manganese concentration in dry, fat-free bone or in bone ash. There were

slight differences in the relationship between sources in Experiment 4

when different response criteria were used. The general relationships,

however, were the sane in all cases. Sources No. 2, 4 and 12 were the

least available in every case. Availabilities ranged from approximately

77 to 97 percent when the effect of the basal diet was included and from

0 to 68 percent when the effect of the basal diet was eliminated.

Solubilities of test materials were in better agreement with

availabilities of these materials in the present experiments than in

Experiments 2 and 3. However, as was found in the previous experiments,









solubility and availability were directly related only in the case of

solubility in neutral ammonium citrate. Sources No. 2, 4 and 12 which

were the least available test materials used in these experiments also

exhibited the lowest solubilities in this solvent. All of the test

sources were essentially insoluble in water.



Summary

Two identical experiments were conducted to determine the avail--

ability of manganese from several inorganic sources of the element. The

response criteria used in the assay were body weight, incidence of leg

abnormalities, percent bone ash, and bone levels of manganese. Reagent-

grade manganese sulfate, used as the reference standard, was added to the

basal semi-purified diet at supplemental manganese levels of 10, 20, 30,

60 and 120 ppm. Six other inorganic sources of manganese were tested

at a level of 10 pp:i added manganese.

A level of 10 ppm supplemental manganese from any of the sources

used was adequate to promote normal growth and bone ash levels. It was

concluded that 20 ppm supplemental manganese from the standard source

was required to prevent perosis entirely. The test sources differed in

their effectiveness in preventing leg abnormalities at the 10 ppm

supplemental manganese level.

Bone manganese level, when expressed as ppm in dry, fat-free bone or

in bone ash, increased significantly with increasing levels of dietary

manganese. U1anganese concentration in the bone increased in a linear

fashion at least up to 30 ppm added dietary manganese. Thus it is con-

cluded that tests for biological availability should be conducted at

supplemental levels below 30 ppm in the area of maximum response.




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