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ABSTRACT

MANGANESE NUTRITION OF THE PIG

BY

Henry B. Kayongo-Male

FOur esperiments involving 104 pigs were conducted to study dif*
ferent aspects of manganese (Mn) nutrition in swine.

In the first experiment, a basal diet (16.2 ppm Mn) and basal
diet supplemented with 10 ppm of Mn from MnSO4'H20, MnCO3 or MnO were
compared for Mn availability to the growing pig. Growth rates were
equal on all diets. Mn availability as measured by Mn balance data and
tissue Mn concentrations indicated that Mn from the supplemented diets
was more available than that from the basal diet. Mn retention, as a
percent of intake, was higher on Mn from the supplemented diets than
the basal diet. Regardless of dietary Mn source, over 90% of excreted
Mn was recovered in the feces. Within the supplemented diets, Mn was
essentially equally available to the growing pig. Hemoglobin, hemato—
crit, serum Mn and serum alkaline phosphatase did not differ signifi—
cantly due to dietary treatment.

In the second experiment, flux patterns across the wall of the
gastrointestine of Mn from different sources was studied. Net absorp-
tion of Mn from the basal diet was evident in two sections of the gut,
the stomach and the cecum, whereas Mn from the supplemented diets was

apparently absorbed in the stomach, cranial small intestine and cecum.

 

 

————-—==——————r”

Henry B. Kayongo-Male
The net cecal absorption of Mn from the basal diet was higher than that
of Mn from the supplemented diets. Net Mn secretion in the caudal
small intestine and the rectum was much higher on the supplemented than
on the basal diet, but this trend was reversed in the colon. The pH
values of the gut contents from different sections of the tract were
not significantly different between dietary treatments.

In the third experiment, two ratios of Ca to P, two levels of Ca

and P and two levels of Mn were studied using a factorial feeding trial.
Mn supplementation significantly increased heart Mn levels and sig-

nificantly depressed rib Ca and Mg values. Mn supplementation did not

 

affect serum Ca, inorganic P, Mg and alkaline phosphatase levels.
Dietary Mn levels had no significant effect on rib and metacarpal
physical measurements, breaking strength and related parameters. A

2 to 1 Ca to P ratio significantly (P<0.05) depressed rib Mn content.
The increased levels of Ca and P supplementation significantly (P<0.01)
increased rib, pancreas and serum Mn levels but significantly (P<0.01)
depressed metacarpal Mn concentration. There was a significnt (P<0.0l
to P<0.05) interaction between levels of Ca and P and ratios of Ca to
P on the levels of serum, liver and pancreas Mn, and on metacarpal Mn
values. High levels of Ca and P in both ratios had a depressing effect
on metacarpal Mn concentration. Feeding Mn along with Ca and P, in a

2 to 1 ratio, increased liver Mn. Metacarpal Mg was depressed when Ca
and P were given in a l to 2 ratio.

The interaction between Ca and P levels and Mn levels was signifi—
cant (P<0.05) with respect to rib ash content, Ca and P, metacarpal Mn
and serum inorganic P. With lower Ca and P levels, Mn supplementation
increased metacarpal Mn and serum inorganic P but depressed rib ash, Ca

and P concentration. The significant effects of Mn supplementation on

 

 

—>—‘_

Henry B. Kayongo-Male
rib Mg, metacarpal internal vertical diameter and heart and serum Mn
disappeared when Ca and P supplements were also fed. The 3—way
interaction between level of Ca and P, ratio of Ca to P and level of
Mn was significant (P<0.05) relative to rib and serum Mn levels,
pancreas dry matter and metacarpal Mg content and elasticity. With
high or low Ca and P levels in a 2 to 1 ratio, Mn supplementation
increased rib and serum Mn and pancreas dry matter but depressed meta-
carpal elasticity. With low Ca and P levels in a l to 2 ratio, Mn

supplementation increased serum and metacarpal Mn, but high Ca and P

 

levels in the same ratio depressed rib and serum Mn, metacarpal Mg
and pancreas dry matter and increased metacarpal elasticity.

Mn supplementation produced more nearly normal histologic struc—
ture of the epiphysis than the basal diet, but animals on high Mn
levels had significantly (P<0.05) less compact bone in the diaphysis.

However, the thickness of the epiphyseal cartilagenous plate was not

 

affected. There was a significant (P<0.05) interaction between Ca to

P ratio and Mn level on the histology of the epiphysis. The interaction
of diet Mn with Ca and P levels was significant (P<0.05) in relation to
the thickness of the epiphyseal cartilagenous plate. These changes

were not typical of rickets but were changes in which there was failure
of production of compact bone in the region of the diaphysis. However,
the deleterious effects on weight gain, feed efficiency and histology

of bone of a low dietary P level (0.35%) from soybean meal were much

more pronounced than the effects of excessive dietary levels of Ca and

P or of an inverse Ca to P ratio, regardless of dietary Mn supplementation.

In the fourth experiment, the Mn requirements of the baby pig born

 

 

0f sows fed a low Mn diet were determined using three dietary Mn concen~

trations. Growth, Mn balance data and serum Mn concentration were used

 

 

Henry B. Kayongo-Male
as measures of sufficiency. Average daily gain, serum Mn, Mn retention,
fecal Mn excretion and urinary Mn excretion as percent of intake were
significantly (P<0.01 or P<0.05) different between dietary treatments.
The average daily gain and feed efficiency were highest on 2.67 ppm Mn
in the diet. Mn intake was highly correlated with serum Mn and serum
alkaline phosphatase activity but not with average daily gain and feed
efficiency. Mn retention as percent of intake had very high negative
correlations with feed efficiency and urinary Mn excretion as percent
of intake. Fecal and urinary Mn excretion as percent of intake was

significantly higher on the basal diet (0.46 ppm Mn). There was a

 

negative Mn retention on this diet. Serum Mg levels substantially
declined in pigs on the basal diet 28 days after the start of the
experiment. Average daily gain was positively related to Mn retention
as percent of intake and negatively related to fecal Mn excretion as
percent of intake. Mn retention and fecal Mn excretion, both as per-

cent of intake, were much more highly correlated with growth rate than

 

absolute Mn intake, excretion and retention and the serum parameters
examined.
Based on all criteria examined, the dietary Mn requirements of the

baby pig on a semipurified diet are probably between 3 and 6 ppm.

 

 

 

 

 

MANGANESE NUTRITION OF THE PIG

By

Henry B. Kayongo—Male

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Husbandry

1974

 

 

 

 

Q0

 

 

1

W

Dedicated to

My Beloved Parents

ii

 

 

 

 

ACKNOWLEDGEMENTS

The author expresses his sincere appreciation to Dr. Ullrey for
his guidance and assistance throughout this work, and for his critical
reading of this manuscript. The writer also wishes to express his
thanks to the other members of his guidance committee, Drs. Miller,
Luecke, and Keahey, for their suggestions and encouragement during
the completion of this study.

I am grateful to the chairman of the Department of Animal
Husbandry and his staff for admitting me to their program. They
provided the necessary facilities unreservedly, which made my stay
comfortable and fruitful.

I must express my sincere thanks to Dr. Magee, whose help with
the statistical analysis will always be remembered, and to fellow
graduate students and laboratory technicians who contributed a great
deal of assistance during the course of this study. Dr. P. K. Ku
and Miss Phyllis Whetter were particularly outstanding. I wish to
thank also Dr. B. W. Wilkinson and Mr. S. Ewald of the Department of
Chemical Engineering for their tremendous assistance, and Miss Janice
Fuller for her skillful preparation of this manuscript.

My parents have been a great inspiration throughout my study

Program. Their encouragement and support have been my motivating

force. I love them.

Most important, I am indebted to my wife Diane, whose sacrifice,

help and encouragement were outstanding.

iii

 

 

 

HENRY B. KAYONGO-MALE
CANDIDATE FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

DISSERTATION: MANGANESE NUTRITION OF THE PIG

OUTLINE OF STUDIES:
Main Area: Animal Nutrition and Husbandry
Minor Areas: Biochemistry
Physiology
Statistics
BIOGRAPHICAL ITEMS:
Born: September 21, 1945, Masaka, Uganda
Undergraduate Studies: Makerere University, Uganda, 1966-1969
Graduate Studies: Michigan State University, 1970-1974

Experience: Graduate Assistant, 1970-1973

MEMBERSHIPS:
American Society of Animal Science

Federation of American Societies of Experimental Biologists
Institute of Nutrition, Michigan State University

iv

 

 

 

 

TABLE OF CONTENTS

LISTOFTABLES..........................Vii

 

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

INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . 4
A. Manganese: An Essential Trace Element . . . . . . . . 4

1. Growth and Development . . . . . . . . . . . . . . 4
2. Reproduction . . . . . . . . . . . . . . . . . . . 10

B. Biological Roles of Mn . . . . . . . . . . . . . . . . l4

1. Enzyme Activation. . . . . . . . . . . . . . . . . 14
2. Energy Metabolism. . . . . . . . . . . . . . . . . 16
3. Hemoglobin Formation . . . . . . . . . . . . . . . 18
4. Ascorbic Acid Synthesis. . . . . . . . . . . . . 19
5. Genetics, Disease and Immunity . . . . . . . . . . 19

C. Mn Metabolism. . . . . . . . . . . . . . . . . . . . . 21

 

1. Absorption . . . . . . . . . . . . . . . . . . . 21
2. Excretion. . . . . . . . . . . . . . . . . . . . 22
3. Retention. . . . . . . . . . . . . . . . . . . . . 24
D. Manganese Requirement. . . . . . . . . . . . . . . . . 25
1. Factors Affecting Mn Requirement . . . . . . . . 25
2. Mn Requirement of Swine. . . . . . . . . . . . . is

3. Mn Requirement of Other Species. . . . . . . . . .
E. Mn Toxicity. . . . . . . . . . . .

EXPERIMENTAL PROCEDURES

0
o
c
o
a
a
o
c
n
I

A. Introduction

B. Experiments.

C.

D.

J-‘wNI—l

0

Experiment 1 . . . . . . . .
Experiment 2 . . . . . . . . .
Experiment 3 . . . . . . . . . . . .
Experiment 4 . . . . . . . . . . . .

Analyses . . . . . . . . . . . . . . . . . . .

1.
2.
3.

Hematological Determinations . . . . . . .
Physical Determinations. . . . . . . . . .
Chemical Determinations. . . . . . . . . .

Statistical Analysis . . . . . . . . . . . . .

RESULTS AND DISCUSSION. . . . . . . . . . . . . . . . . .

A.

D.

Experiment 1 . . . . . . . . . . . . . . . . .

In vitro Solubility of Manganese Compounds .

Mn Availability Studies Using a Split-Plot
Design . . . . . . . . . . . . . . . . . .
Mn Availability Studies Using a Replicated
Latin Square Design. . . . . . . . . . . .
Discussion of the Results of Experiment 1

Experiment 2 . . . . . . . . . . . . . . . . .

Experiment 3 . . . . . . . . . . . . . . . . .

l.

Histopathology . . . . . . . . . . . . . .

Experiment 4 . . . . . . . . . . . . . . . . .

CONCLUSIONS . .

BIBLIOGRAPHY.

APPENDIX.

vi

 

43
43
43
45
52
53
53
53
54

64
68

7O

83

94

97

119

121

139

 

 

 

 

 

 

LIST OF TABLES
Lble Page

1 Diets used in Experiment 1 . . . . . . . . . . . . . . . . 35

 

2 Diets used in feeding trial (Experiment 3) . . . . . . . . 39
3 Low manganese gestation diet (Experiment 4). . . . . . . . 41
4 Purified diets used in Experiment 4. . . . . . . . . . . . 42

5 Chemical composition and solubility of different
manganese compounds. . . . . . . . . . . . . . . . . . . . 53

 

6 Analysis of variance for Mn source effects on bone and
blood parameters (split-plot design) . . . . . . . . . . . 55

7 Analysis of variance for Mn source effects on Mn balance,
tissue Mn concentrations, organ weights and growth data
(split—plot design). . . . . . . . . . . . . . . . . . . . 56

8 The effect of Mn source on the physical characteristics

of different organs. . . . . . . . . . . . . . . . . . . . 57
9 The effect of Mn source on the physical measurements of

the femurs and ribs, breaking strength and related

parameters of the femurs . . . . . . . . . . . . . . . . . 58

3 The effect of Mn source on the average daily gain (ADC)
and tissue Mn distribution . . . . . . . . . . . . . 59

L The effect of Mn source and time of sampling on the
blood and balance data (split-plot design) . . . . . . . . 61

3 Effect of Mn source and time of sampling on some blood
parameters . . . . . . . . . . . . . . . . . . . . . . . 62

3 Retention and routes of excretion of Mn from basal diet
and basal diet supplemented with manganous sulfate
(MnSO4.H20), manganous carbonate (MnC03) or manganous
oxide(MnO)........................ 63

l Effects of Mn source on blood and Mn balance measures

(replicated Latin square design) . . . . . . . . . . . . . 65

3 Effect of Mn from different sources on some blood

. . . . . . . . 66
measurements . . . . . . . . . . . .

vii

 

 

 

Lble Page

fl Retention and routes of excretion of Mn from basal diet
and basal diet supplemented with manganous sulfate
(MnSO4~H20), manganous carbonate (MnCO3) and manganous
oxide (MnO). . . . . . _

. . . . . . . . . . . . . . . . . . 67

J Net absorption and secretion of Mn in the different
sections of the gut by the growing pig fed different

 

Mn sources . . . . . . . . . . . . . . . . . . . . . . . . 71
8 Cut content weights and pH . . . . . . . . . . . . . . . . 72
9 Mean, minimum and maximum values of pH, wet gut contents

and net absorption and secretion in different sections
of the gut . . . . . . . . . . . . . . . . . . . . . . . . 73

0 Effect of the Ca-P ratio, Ca and P levels and Mn sup-
plementation on physical and chemical parameters of the
first, left rib. . . . . . . . . . . . . . . . . . . . . . 85

1 Effect of the Ca-P ratio, Ca and P levels and Mn sup-
plementation on physical, chemical and histopathological
parameters of the 5th metacarpal . . . . . . . . . . . . . 86

 

2 Effect of the Ca—P ratio, Ca and P levels and Mn sup—
plementation on organ and blood composition. . . . . . . . 87

3 Effect of the interaction between Ca—P ration and level
of Ca and P on Mn metabolism and some other bone
parameters . . . . . . . . . . . . . . . . . . . . . . . . 89

4 Effect of the interaction between Ca—P ratio and level
of Ca and P on organ and serum Mn metabolism, and some
other measures . . . . . . . . . . . . . . . . . . . . . . 90

3 Effect of the interaction between Ca—P ratio and Mn
levels on Mn and Mg metabolism . . . . . . . . . . . . . . 92

5 Effect of the interaction between Ca—P levels and Mn
levels on some physical parameters . . . . . . . . . . . . 93

7 Effect of the interaction between Ca—P ratio, Ca and P
level and Mn level on some measured parameters . . . . . . 95

3 Analysis of variance of effect of dietary Mn level on

growth, balance and blood measures . . . . . . . . . . . 98

9 The effect of dietary Mn levels on serum and growth 100
parameters . . . . . . . . . . . . . . . . . . . . . . . .

) Retention and excretion routes of Mn from diets con— 102
taining different levels of Mn . . . . . . . . . . . . .
Correlations between Mn balance measures, serum 104

parameters and growth. . . . . . . . . .

viii

 

 

Table Page

32 Correlations within the serum parameters . . . . . . . . . 106
33 Correlations within the Mn balance measures. . . . . . . . 107
A-l Michigan State University vitamin—trace mineral premix . . 139

A-2 Mineral mixture used in semi—purified experimental diets . 139
A—3 Vitamin mixture used in semi—purified experimental diets . 140
A—4 Cleaning polyethylene vials. . . . . . . . . . . . . . . . 140

A—5 IL Model 335 flameless sampler . . .

 

 

 

 

LIST OF FIGURES
ure Page

1 Summary of net Mn flux from sections of the gastro-
intestinal tract of the growing pig fed the basal
diet . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

2 Summary of net Mn flux from sections of the gastro—
intestinal tract of the growing pig fed the basal
diet supplemented with MnSO4'H20 . . . . . . . . . . . . . 77

3 Summary of net Mn flux from sections of the gastro—
intestinal tract of the growing pig fed the basal
diet supplemented with MnCO3 . . . . . . . . . . . . . . . 79

4 Summary of net Mn flux from sections of the gastro—
intestinal tract of the growing pig fed the basal
diet supplemented with MnO . . . . . . . . . . . . . . . . 81

l The effect of dietary Mn concentration on serum Mg
concentration. . . . . . . . . . . . . . . . . . . . . . . 110

Z The effect of dietary Mn concentration on serum
alkaline phosphatase activity. . . . . . . . . . . . . . . 112

3 The effect of dietary Mn concentration on serum Mn
concentration. . . . . . . . . . . . . . . . . . . . . . . 114

The effect of dietary Mn concentration on average
daily gain (ADC) and feed efficiency (F/G) . . . . . . . . 116

Mn balance data, intake, fecal and urinary excretion
and retention on different dietary Mn levels . . . . . . . 118

 

 

 

 

 

INTRODUCTION

Nutritional studies with manganese (Mn) were stimulated by the
iscovery that two poultry diseases, perosis and chondrodystrophy, were
aused by inadequate intakes of Mn and could be prevented by Mn sup—
1ementation. Since then Mn has been assigned a growing number of
iological functions vitally important to the organism. This element

3 now associated with normal activation of enzymes and co-enzymes

 

ssociated with feed utilization. Manganese plays an important role
n liver function, bile production, energy metabolism, reproduction and
roper skeletal development. The occurrence of Mn in all tissues and
be small range of Mn concentrations indicate a role of this element
general cell metabolism as opposed to metabolic processes character—
tic of particular tissues.
Although Mn is an indispensable micro-element of considerable
actical importance in animal feeding, its metabolism in swine has
d limited research, with little attention to its availability, site
absorption and requirement at different stages of development.

nganese interactions with other elements and vitamins, shown in other

 

ecies, have not been studied in swine.

Manganese compounds have different chemical and physical properties
ich are important in determining their availability to animals. Since
e cost of Mn compounds varies widely, it is important not only to

fine accurately the nutritive requirement of Mn for swine at

 

 

 

 

 

 

 

2

ferent stages of their development but also to define the less
ensive but equally effective ingredients to use in the mineral
mix.

The Mn flux pattern across the gastrointestinal mucosa in swine
not been extensively studied. Such influx-efflux studies can
vide information on the extent of absorption of the mineral and
secretion, in various sections of the digestive tract.

Manganese interactions with Ca and P have been studied in some
ail in poultry and laboratory animals, but most of those findings
3 not been examined in swine. Interactions of this element with
er elements, such as copper and iron, and vitamins have not been
iied in swine. Verification of any Mn interactions in swine is
thant not only to the understanding of the utilization of Mn but
3 of other elements in the presence of low or high dietary levels
in.

The National Research Council has suggested 20 ppm Mn as the
irement for a young growing pig. Manganese requirements that have

reported in the literature vary widely. There is a definite need
efine accurately the Mn requirement of the baby pig. Modern manage-
: practices, increased knowledge of other nutrient needs and ever-
Lging breeding programs in the swine industry all call for reevalua-
[Of Mn requirement data. The development of more sensitive and
:ific analytical methods also necessitates this reexamination.

The experiments to be reported in this dissertation were designed
tudy the following aspects:

1. The availability of Mn from different Mn sources for the

'ing pig.

 

 

 

 

 

 

 

 

 

 

3
2. The flux pattern of Mn across the gastrointestinal tract
in the pig.
3. The effect of high level supplementation of Ca and P and a
Za-P ratio less than 1.0 on Mn utilization.

4. The Mn requirement of the baby pig born of a Mn-deficient sow.

 

 

 

 

 

 

 

 

 

 

LITERATURE REVIEW

Manganese: An Essential Trace Element

 

1. Growth and Development

 

The first experiments with rats and mice attempting to demon~

ate the essentiality of Mn using purified diets were suggestive but

 

conclusive. In 1931 Kemmerer et al. found better growth rates of

 

 

nle mice when 0.1 mg of Mn was added daily to a milk diet containing

 

.tional Fe and Cu. Since that time many workers have demonstrated
essentiality of Mn for optimal growth in rats. Boyer et a2. (1942)

vrted that young rats from Mn-deficient dams showed a markedly

‘er growth rate and poorer feed utilization than similar rats on the
diet supplemented with Mn. Randoin (1944) found similar subnormal
th, followed by a rapid decline and death. Holtkamp et a2. (1950)

ed that a subcutaneous injection of colloidal Mn improved growth

. Supplements of Mn improved growth rate and feed efficiency in

aa pigs and rabbits (Everson, 1970; Ellis et al., 1947). Rabbits

a Mn-free diet showed signs of anorexia (Rudra, 1944a) and, when

on this diet for a long time, the animals ceased to grow normally
lied. Postmortem examination revealed hemorrhages of the lungs,

: and intestinal capillaries.

Wachtel et a2. (1943) reported that Mn deficiency in rats caused
bone formation and Frost et a2. (1959) observed delayed skeletal
'ation in rats on Mn-deficient diets. O'Dell (1961) described short

4

 

 

 

 

 

5

ed forelegs, extra sternebrae and fusion of the sternal and vertebral
ents of Mn-deficient rats. Hurley at al. (19613) confirmed the
rtening of the long bones, both absolutely and relative to body

gth, and reported that the radii and tibia were greatly thickened
distorted; longitudinal growth of the skull was reduced, and the

th and height of the skull were slightly less in the deficient

mals on an absolute basis, but in proportion to length of the skull

y were greater. They also reported that ribs were either missing or

ormed and the chest was anteriorly—posteriorly flattened. Abnormal
e development due to Mn deficiency has been described in rabbits by
th at al. (1944) and Ellis at al. (1947). Only the front legs were

d; the ulna and the humerus were significantly reduced in size,
sity, Mn content and breaking strength. X—ray and microscopic
dies showed changes distinctly different than those seen in rickets.
narrowing of the zone of provisional calcification and the epiphyseal
te, and a deficiency of spongy bone indicated a suppression of
aogenesis (Smith at al., 1944)-

Concrete evidence that Mn is involved in bone formation was
Lined by Parker et a2. (1955), who showed that a greater concentra—
L of injected radioactive Mn was found in the regions of greater
2 formation, by Leach (1967), who showed that the changes in epiphyseal
wilage of growing bone of Mn-deficient chick embryos were due to a
ction in mucopolysaccharide production, and by Tsai and Everson

7), who reported a similar reduction in mucopolysaccharides due to
duced hexose utilization for their synthesis in Mn-deficient guinea
. The conclusion by Caskey et a1. (1939) that abnormal bone develop—
was probably the result of disturbance in Ca and P metabolism in

absence of Mn was disproved by Parker at al. (1955), who showed that

 

 

 

 

 

 

 

 

 

6

e quantities of radioactive Ca and P deposited in bone were not
fected by Mn intake.

Shils and McCollum (1943) reported ataxia, incoordination and poor
uilibrium in young rats from Mn-deficient dams. Hurley and Everson
959) and Hurley at al. (1961b) found that the offspring of Mn-deprived
thers were slower to acquire reflexes and showed inadequate develop-
nt of the bony labyrinth. Hurley at al. (1961b) found that the brain
ights of Mn—deficient rats were absolutely smaller, but relative to
e body weight, larger than the controls; the cerebrospinal fluid
essure did not differ significantly. Assays of tissues for a number
enzymes, including acetylcholine esterase in the brain of Mn,
ficient rats and guinea pigs did not reveal any biochemical faults
an Reen and Pearson, 1955). Shrader and Everson (1967) and Hurley
969) concluded that abnormal development of the otoliths was
sponsible for the congenital ataxia present in rats and guinea pigs
en the maternal diets were Mn-deficient. Shrader and Everson (1967)
ported abnormal curvatures of the semi-circular canals and misshapen
pullae in Mn—deficient guinea pigs. However, hematoxylin and eosin
:tions of the vestibular apparatus of ataxic rats failed to show
ysical lesions (Hill and Holtkamp, 1954). Erway at al. (1970) showed
at the incidence of ataxia increased in proportion to the severity
1 duration of the Mn deprivation of the mother.

There is a depressed growth rate in chickens on low dietary Mn
illup and Norris, 1939a; Litricin, 1967). Improved growth rates due
Mn supplementation of low Mn basal diets have been reported by
:tle at al. (1969), Belincenko (1968), and Van Reen and Pearson
’55). Since Wilgus (1936, 1937) showed that addition of 25 ppm of

to a basal diet containing 10 ppm of Mn completely prevented perosis,

 

 

 

 

 

 

7

ier workers have confirmed this finding (Underwood, 1971). The
urrence of perosis on low Mn diets has been reported in ducklings
in Reen and Pearson, 1955) and in turkey poults (Vohra and Heil,
59). Wolbach and Hegsted (1953) found a suppression of the epiphyseal
:tilage developmental sequence, with immature cartilage cells, fol-
7ed by retarded tunnelling and abnormal matrix in the zone of growth.
[ch (1968) reported a reduction in width of epiphyseal plate and
:aphysis. Creek at al. (1960) reported that bone deformities of the
:k'joint in perosis were aggravated by weight applied to the leg.

Lyons and Insko (1937) reported that nutritional chondrodystrophy
caused by inadequate intakes of Mn, and Litricin and Andrejevic
66) found that in Mn deficiency the long bones of the legs and wings
e significantly smaller, had reduced diameter and length but did not
cken. Caskey and Norris (1940) did not find any significant effect
the sternum and metacarpus due to dietary Mn levels. Ataxia in the
spring of Mn—deficient chickens was first observed by Caskey and
ris (1940). Caskey at al. (1944) showed that ataxic chicks from
deficient parents grew normally on Mn-supplemented diets. They
cribed the ataxia as a tetanic spasm of the opisthotonic type.
tologically, the brain was normal, but chemical changes in lipid,
al phosphorus and phosphatase content were noted.

Manganese is involved in egg shell formation. Pullets fed rations
:aining low Mn with high levels of Ca and P produced eggs with
arior shell characteristics (Lyons, 1939; Longstaff and Hill, 1971).
>wska and Parkhurst (1942) showed that feeding chickens Mn-deficient
:s significantly reduced the eggshell breaking strength, but Chubb
54) found no detectable difference in eggshell quality due to Mn

>1ementation. Recently Hill and Mathers (1968a), Mathers et a2.

 

 

 

 

 

 

 

 

8

(1971) and Longstaff and Hill (1970) have shown that eggshell thickness
as depressed by a low Mn diet when given before laying but not when
iven from the point of lay. They also showed that low Mn diets
epressed significantly the acid mucopolysaccharide content of the
shell matrix.

The nutritional significance of Mn in ruminant feeding has not
>een clearly verified. Bentley and Phillips (1951a) showed that heifers
fed diets containing 7 to 10 ppm Mn were slower to mature, and more of

:heir calves were born with weak legs and pasterns at first calving.

 

:rashuis at al. (1953) has reported leg deformities with overknuckling

Lnd poor growth in calves of cows grazing Mn depleted pastures. Rejas

at al. (1965) reported reduced breaking strength and length of the

Lumerus of deformed calves from Mn—deficient cows. Hartmans (1970)

id not find any Mn deficiency symptoms in grazing animals in Holland.

nke and Groppel (1970) reported that there were no growth differences

n female goats fed a ration containing 20 ppm Mn in the first year and
ppm in the second year as compared to the controls consuming a similar
ation supplemented up to 100 ppm of Mn. Lassiter and MOrton (1968)

ave reported that Mn—deficient sheep developed poor bones with low

sh concentration in the femur and reduced Ca, P and Mn content in the

 

sh, implying an effect of Mn on bone ossification.

Manganese deficiency has been shown to cause poor growth and lame-
ess in growing swine. Miller at al. (1940) reported the occurrence
f lameness in pigs at 27 kg when they were fed high-corn diets contain-
ng 14 ppm Mn. The condition was characterized by a slight halting
ait, progressing into enlarged hooks and crooked legs, becoming painful
Jr the pigs to rise to their feet. Bone analysis of the Mn—deficient

[gs showed normal mineral deposition. Higher Mn supplementation did

 

 

 

 

 

 

9

ct cure the stiffness but did prevent it. This work was confirmed by
eith at al. (1942), but in addition they showed that Mn—deficient pigs
ere non-ataxic and had good appetites. Sandstedt and Carlquist (1951)
bowed similar bone deformities, characterized by swollen streaks on
the posterior external contours of the hooks in pigs fed Mn-deficient
iiets. Johnson (1943) obtained satisfactory growth rate and no early
lameness on purified diets containing less than 0.5 ppm of Mn. However,
after long periods of time on this diet some signs of lameness appeared.

Leibholz at al. (1962) found that 0.4 ppm Mn in the diet of baby pigs

 

was sufficient to support maximum growth. Johnson (1944) reported
satisfactory growth rates from weaning to market weight on low Mn
liets, and Mn supplementation was only slightly beneficial when the
Lsh content of the ration was raised above 10 percent. Giessler and
drchgessner (1959) found that Mn supplementation did not improve
eight gains, feed intake or efficiency of conversion of Swabian—Hall
igs fattened from 20 to 96 kg on a diet of barley with 250 g of a
tandard German protein concentrate daily. But Williams and Noland
1949) found that Mn supplementation, along with other trace elements,
mproved both the growth rate and feed efficiency in swine.

Grummer et a1. (1950) fed pigs a corn-soy basal diet containing 12

 

pm Mn supplemented with 40, 80, and 160 ppm Mn. Pigs consuming diets
upplemented with 40 ppm gained significantly faster than those on the
asal diet but performance of the pigs was not improved with higher
evels of Mn. There was a slight depression of bone ash of the pigs on
he basal diets. Plumlee et al. (1956) showed no significant difference
a growth rate and feed efficiency between groups of Duroc boars fed

1 diets ranging from 0.5 to 40 ppm Mn. Pigs on the low Mn diets showed

tendency towards sickle—hocks; and X—ray photographs of the legs of

 

 

 

 

 

 

10
-deficient gilts showed that growth of the radii was prevented by
:sure of the distal epiphyseal plate. Plumlee at al. (1956), feeding
.ts from.Mn-deficient sows on Mn-deficient diets, observed signs of
deficiency manifested by pain and weakness of the legs at 27 kg
reweight, and shortened and thickened front legs becoming bowed at
kg. The deficient pigs were short in the body and excessively fat.
Ler at al. (1956) reported similar defects in young pigs on purified
:ts of low Mn content. They showed generalized rarefaction of leg

LES, but the lameness and rarefaction disappeared after 335 days on

 

~erimental diets, whereas the other signs of Mn deficiency persisted.

2. Reproduction

 

Kemmerer et al. (1931) reported that mice reared on cows’ milk
plemented with Fe and Cu failed to ovulate normally, but mice on the
e diet with 0.01 mg of Mn added daily exhibited normal estrous cycles.
nt and McCollum (1931) fed young rats on Mn-free diets; the females
ibited normal estrous cycles, but all their litters died, apparently

to deficient lactation. The males displayed testicular abnormali—

s. Boyer at al. (1942) reported irregular or no estrous cycles in

 

ale rats raised on low Mn diets, and there was a marked delay in the
ning of the vaginal orifice; the males had testicular degeneration
complete sterility owing to lack of spermatozoa production when fed
imilar diet. Shils and McCollum (1943) reported that when Mn—
icient females were mated to normal males, only 1.5 percent of the
mg survived to weaning as compared to 56 percent of the supplemented
1p. Male rats on low Mn diets from weaning did not show differences
3Perm mortality and testicular weight. There were increased still—

:hs and depressed survival rates when male and female Mn-deficient

 

 

 

 

 

 

ll
lbino rats were mated (Barnes et aZ., 1941). Hurley et a1. (1958)
onfirmed these effects and reported that they were aggravated in
ubsequent generations. Smith et al. (1944) and Ellis at al. (1947)
130 reported testicular degeneration and lack of libido in male
abbits on low Mn rations, and reduction in size of ovaries and uterii
n females. In guinea pigs, the omission of Mn from the maternal diet
esulted in a decrease in litter size and an increase in percentage of
oung born prematurely or ataxic or dead (Everson at aZ.. 1959).

Gallup and Norris (1939b) showed that Mn deficiency in the diet

 

f chickens resulted in decreased egg production, fertility and hatcha-
ility. Others have reported improved reproductive performance when
asal diets were supplemented with Mn (Underwood, 1971; Atkinson 9t

1., 1967). Christiansen at al. (1940) reported that Mn and flavin

are the critical factors involved in the subnormal hatchability associ—
:ed with soybeans. Hoogendorn (1940) found that with birds kept

idoors, the addition of Mn to the diet improved egg production and
itchability. Gutowska and Parkhurst (1942) and Hill and Mathers

.968b) did not find any improvement in egg production, fertility and

 

Ltchability due to Mn supplementation. Chubb (1954) found that pullets
ed on low Mn diets produced twice as many infertile eggs and dead—in—
Lell embryos as those on 50 ppm Mn diets. An increased incidence of
:ad retractions in newly hatched chicks and chondrodystrophic embryos
5 associated with lines of birds producing eggs of low Mn content
olton, 1957).

Bentley and Phillips (1951a) showed that Mn-supplemented Holstein
1ves came into heat earlier than those on low Mn diets, suggesting a
imulating effect of Mn on sexual maturity; the number of services per

nception and number of calves born and calves born dead were unchanged

 

 

 

 

 

 

 

12

 

y dietary treatments, but the calves born of Mn-supplemented cows in
be second generation were noticeably heavier at birth. Experimental
nd field data show that Mn is necessary for fecundity in cattle
(Rojas at al., 1965; Bentley at aZ.. 1951). Bentley et al. (1951)
ound a significantly lower Mn content in the ovaries of repeat-
reeders, but there was no direct relationship between the content of
in feed, tissue and organs, and infertility. Werner and Anke (1960)
bowed a relationship between the Mn supply and number of services per
onception. Dyer (1960, 1961) and Dyer at al. (1964) reported a positive
elationship between a low Mn intake by gestating cows and the incidence
f neonatal deformities in their calves. Pastures with 15 ppm Mn or
JElOW resulted in greater incidence of infertility and abortion.
5onomi (1966) and Wilson (1966) reported that high dietary levels of
:a0 and P205 seemed to cause Mn deficiency, which precipitated func—
Lional infertility in cattle. Manganese supplementation has been shown
0 improve the fertility of cattle in Europe (Grashuis et aZ.. 1953;
ilson, 1965, 1966; Krolak, 1968). Anke and Groppel (1970) found that
oats on low Mn diets came into estrus late, the symptoms of estrus were
eak, and more inseminations per conception were needed. The low Mn
roup produced more male kids but also had more abortions. Similar
bservations were reported in guinea pigs by Everson at al. (1959).
Manganese involvement in reproductive processes of cattle was
urther proven by Anke and Groppel (1970), who showed a higher concen—
ration of the element in the ovaries after an injection of radioactive
n, and in Archibald and Lindquist (1943), who showed that there was a
ubstantial transfer of Mn into the milk by the ovine mammary gland.
Johnson (1940) reported that bred sows fed a semi—purified diet

antaining 0.3 ppm Mn produced apparently normal pigs; however, the pigs

 

 

 

 

 

 

 

 

13
contained one tenth as much Mn as those fed 6.0 ppm Mn. Sows fed diets
containing 6.0 ppm Mn raised their litters satisfactorily. Johnson
(1944) showed satisfactory reproduction through two generations on a
ration of 8.6 ppm Mn and 1.78 percent ash. Grummer et al. (1950)
reported that sows on diets containing 12 ppm Mn tended to be less
fertile, hard to settle and gave birth to smaller and abnormal litters.
Supplementation of the basal diets with 40, 80, or 160 ppm.Mn for sows
during reproduction and lactation resulted in a significant difference
in performance of their litters. Gligor at al. (1966) reported no sig-
nificant effect on weight of sows at farrowing or weaning or 60 days
later, or on number and weight of piglets. Speer et al. (1952) showed
that pigs from sows supplemented with 70 to 90 ppm Mn during gestation
and lactation made highest gains during the growing and fattening
periods. Plumlee et a1. (1956) fed female swine either low or high Mn
iiets throughout growth, gestation and lactation, and reported that the
growth rate and feed efficiency of their litters were satisfactory at
30th levels. In another experiment, Plumlee at al. (1956) reported that
when female pigs were fed on either low or high Mn diets, the differ-
ances in number of pigs farrowed or viability or birth weights of
Litters could not be attributed to Mn levels given. But when gilts
rere depleted of Mn by feeding Mn-free purified diets, the reproductive
>rgans were histologically normal, and they ovulated normally but
:howed irregular estrus and sometimes would not accept the boar. When
mted, they farrowed weak ataxic litters, unable to suck, and most of
:hem died even when transferred to Mn-supplemented diets. The litters
rom supplemented gilts were normal, vigorous and healthy. Boars from
ilts getting either low or high Mn levels in their diets, all reared on

3.3 ppm Mn ration, performed equally well and showed normal

 

 

 

 

 

 

 

 

l4
ermatogenesis (Plumlee et al., 1956). Neher at al. (1956) reported
at sows on a low Mn diet farrowed abnormal pigs with reduced birth—
ights. The above findings were not sustained by the results of
ibholz at al. (1962) and Newland and Davis (1961), who found,
dependently, that sows on lOW'Mn diets farrowed or produced normal
tuses without apparent reduction in birthweights. The involvement
Mn in swine reproduction is further shown by the findings that there
a rapid, unlimited transfer of the element to the fetus, colostrum
1 milk (Plumlee et al., 1956; Newland and Davis, 1961; Leibholz at

., 1962).

Biological Roles of Mn

 

1. Enzyme Activation

 

Lehninger (1970) lists many enzymes involved in intermediary
:abolism that require Mn ions for activation. Mn is an integral part
many enzyme systems of the body. Some of the specific enzymes for
.ch Mn is known to be essential as an activator are acetyl CoA
“boxylase (Wells and Remy, 1965), cytochrome oxidase (Vorob'eva,

0), enolase (Wacker at al., 1964; Babin et al., 1964): frUCtOSE 1:6
hosphatase (Pontromoli at aZ., 1969), heparinase (Dietrich at al..
2) and succinic dehydrogenase (Babin at al., 1964)- Other divalent
ions, especially Mg, with similar properties, can replace Mn in the
ivation of many enzyme systems.

Rubenstein at al. (1962) showed that high levels of Mn induced

are hypoglycemia due to increased enzyme activation, and Johnson at
(1959) reported that preincubation with Mn ions increased consider
I the proteolytic activity of rat and chick pancreatic homogenates.

:terjee et al. (1960) found that Mn ions restored the conversion of

 

 

 

 

15

;1uconate to ascorbic acid by goat liver microsomal enzymes. Skinner
Lnd McHargue (1944) found that Mn supplementation of rats increased
o-carboxylase activity. Reineke and Turner (1945) showed that enzymes
.nvolved in the iodination of tyrosine to diiodotyrosin and subsequent
~xidation to thyroxine were best catalyzed by Mn, especially colloidal
moz. Van Reen and Pearson (1955), studying a number of enzymes in
n—deficient and Mn-supplemented ducks, showed that Mn had no effect

n the activity of liver diphospho-pyridine nucleotidase, cytochrome

xidase, catalase and isocitrate dehydrogenase. Leach (1967) found that

 

he enzymes, polymerase and galactotransferase, involved in the chon-
roitin sulfate synthetic system are activated by Mn.
In vitro studies indicated that Mn ions activated phosphatase, but

ddition of Mn ions to an enzyme preparation from the bones and blood
f a perotic chick did not raise the activity to nearly the same level
5 that found for a chick without perosis (Weiss at al., 1939). Wachtel
t al. (1943) found that Mn deficiency caused a significant increase in
lood serum.phosphatase but not bone phosphatase. But Ellis et a1.
L947), Leibholz et a2. (1962) and Rojas et a2. (1965) showed a depres-
Lon of bone alkaline phosphatase activity on low Mn diets. Combs at
l. (1942) found an intimate relationship between the phosphatase
:tivity of the bones and Mn deficiency. Lowering of the phosphatase
:tivity retarded bone development. Phosphatase activity was greatly
duced by withdrawing Mn. Lassiter et a2. (1970) reported that sub—
rmal bone alkaline phosphatase activity does not invariably occur with

deficiency. Hurley and Everson (1959) showed that both Mn-deficient
ts and controls showed the same pattern of enzyme activity and there
re no significant differences between them. Nielsen and Madsen (1942)

owed that blood phosphatase significantly increased in perotic

 

 

 

 

l6

rkeys, but Van Reen and Pearson (1959) found that the enzyme activity
livers of Mn—deficient ducks was only one—half that of the livers of
pplemented birds. Lassiter et al. (1970) found that serum alkaline
osphatase activity of lambs fed 1 ppm Mn was significantly below that
' controls receiving 29 ppm Mn, but kidney alkaline phosphatase
:tivity was increased. Leibholz et a1. (1962) showed that the alkaline
msphatase activities of the kidney, liver and serum of baby pigs given
4 ppm Mn in the diet were not affected.

' Boyer at al. (1942), Rehner and Stelte (1970) and Rehner and Cremer
.970) reported the arginase activity of liver preparations from Mn—
ficient animals was greatly increased by Mn additions. Mn supplementa-
.on increased urea in blood, urine and saliva of cattle (Zerebeov at
,,, 1970; Rozybakiev, 1966). Others have shown a depression in
ginase activity of rats and guinea pigs on Mn-deficient diets (Ellis

aZ., 1947; Everson, 1970). But Leibholz at al. (1962) found that Mn
eatments did not significantly affect the liver and kidney arginase

tivity in baby pigs.

2. Energy Metabolism

 

Lehninger (1970) lists a number of key enzymes involved in the

rcolytic pathway, gluconeogenesis and beta-oxidation requiring Mn

[8 for activation. Early investigations by Ray and Deysach (1942)
>wed that subcutaneous injection of Mn into guinea pigs in small

as raised oxygen consumption, but higher doses up to 100 mg per kg
bodyweight progressively depressed oxygen consumption. wachtel at
(1943) found no Mn effect on basal metabolic rate (BMR) in rats.

Bentley at al. (1951b) found that phosphorylation of chicken liver

>genates was increased 41 percent by Mn above that on a choline—

 

 

 

 

 

 

 

17

:ficient, Mn-deficient ration. Hurley et a2. (1970) showed a depres—
.on in oxidative phosphorylation in Mn-deficient mice. Buccellato
953) found in vitro that a compound formed between pyridoxine and
lloidal Mn had an active role in carbohydrate metabolism. Scrutton
a2. (1966) found that pyruvate carboxylase is a Mn metalloprotein,
d Mildvan at al. (1966) showed that Mn functions in the transcarboxyla—
on part of the pyruvate carboxylase reaction.

Manganese involvement in glucose utilization was shown by Everson
i Shrader (1968) and Shrader and Everson (1968). Newborn guinea pigs,
verely deficient in Mn, exhibited a marked hypoplasia of all cellular

nponents of the pancreas, with fewer and less intensely granulated

 

:a cells than the controls. When glucose was administered orally or

:ravenously to young adults which were congenitally Mn—deficient,

 

:y showed glucose responses resembling the diabetic subject, whereas
[trol animals always returned promptly to normal glucose levels. Mn
bplementation of deficient animals completely reversed the reduced
.cose utilization in guinea pigs and cattle (Everson and Shrader,
»8; Zerebeov at al., 1970). The administration of this element to
betic subjects has a hypoglycemic effect (Mehrolera at al., 1964;
enstein, 1962); and both pancreatectomy and diabetes have been
elated with decreased Mn levels in blood and tissues (Konseko,
).
Manganese supplementation was shown to reduce liver and bone fat
-deficient rats (Amdur at aZ., 1946). Plumlee et a1. (1956) showed
Mn-deficient gilts were excessively fat by 25 to 40 kg liveweight.
an (1954) showed that Mn stimulates the hepatic synthesis of
esterol and fatty acids in rats. Mn ions are necessary for the

ersion of mevalonic acid to squalene by mevalonic kinase (Amdur

 

 

 

 

 

18

al., 1957); and the phosphorylated derivative of mevalonic acid,
essary for this reaction, requires Mn for its synthesis. Barron

66) showed that Mn was a necessary co—factor for mitochondrial fatty
d synthesis together with NAD and citrate; and others have reported

t Mn inhibits lipoamide dehydrogenase (Lehninger, 1970).

3. Hemgglobin Formation
Manganese has been shown to either increase or decrease or
e no effect on hemoglobin values in a variety of animal species.
htel at al. (1943) and Smith at al. (1944) found that hemoglobin
els were not significantly affected by lack of Mn. But Skinner and
rgue (1946a), using dry or milk diets, showed that Fe, Cu and Mn
e higher hemoglobin values than Fe and Cu added alone.

High levels of Mn (100 to 3000 ppm) had a small but significant
ression on hemoglobin levels of calves (Cunningham at al., 1966).
oglobin regeneration was greatly retarded and serum iron depressed
anemic and normal lambs, rabbits and pigs by feeding high levels of

The levels causing reduction of hemoglobin ranged from 50 to 5000

(Robinson at al., 1960; Matrone at al., 1959; Hartman at aZ.,

5). With baby pigs, Matrone et a2. (1959) showed that the regenera—
1 of hemoglobin, when Fe intake was low, was depressed by excess Mn
ikes but the depression was overcome by extra Fe. When the anemic

7 pigs, given 25 ppm Fe and 5 ppm Cu in the diet, were supplemented
1 125, 250 or 2000 ppm Mn, all the levels of Mn depressed hemoglobin
nation and the drop was sharp and significant. Mbinuddin and Lee
50) found a decline in hemoglobin concentration, a reduction in red
Dd cell count and an increase in white blood cell count due to feed-

high Mn levels. In 1960, Sullivan noted similar changes in rats

 

 

 

 

 

 

 

 

19

van a manganese edetate supplement. The changes were greater in

ung rats.

4. Ascorbic Acid Synthesis
Rudra (1939) found that rat and guinea pig livers were able to
thesize ascorbic acid when incubated with mannose or galactose in
e presence of Mn which acts as a co-enzyme in the conversion. Later
showed that intraperitoneal injection of 20 mg of mannose in the
esence of 0.04 percent Mn to guinea pigs gave a small increase of
orbic acid in body tissues and protected against scurvy (Rudra,
44b). He concluded that Mn was essential for the synthesis of vitamin
in animals and that failure to synthesize it is due to insufficiency
the metal at the site of ascorbic acid synthesis.
Injections of mannose plus Mn given to scorbutic guinea pigs did
t stimulate ascorbic acid synthesis from mannose in viva (Skinner and
iargue, 1946b). The Mn involvement in ascorbic acid synthesis is
nplicated by vitamin E (Caputto at aZ., 1958). Using enzyme prepara—
>ns from vitamin E—deficient rats, 70 to 90 percent less ascorbic
d was produced than from a preparation taken from animals given
ficient vitamin E, and addition of Mn to in vitro systems increased
ascorbic acid produced by vitamin E-deficient preparation 315

cent.

5. Genetics, Disease and Immunity
A difference in Mn requirement to prevent perosis among various
eds of birds has been documented (Golding at aZ., 1940; Pills,
8). Caskey at al. (1944) reported that offspring of ataxic female
male chickens grew normally on a diet supplemented with Mn, sug—

ting the ataxia was not complicated by the inheritance of a simple

 

 

 

 

 

 

 

20

ecessive character influencing Mn retention. Hurley (1969) showed that
taxia caused by maternal Mn deficiency is indistinguishable from.that
used by certain genes in mutant mice. A supplement of 1000 ppm.Mn
pregnant females on a diet that normally produced 68 percent ataxic
ung completely prevented the condition and, when the normal offspring
re mated and fed a normal diet, the normal offspring subsequently
oduced 68 percent ataxic young. Thus, the high level of Mn prevented
ression of the genetic abnormality without influencing genetic
nstitution.
Hoogendoorn (1940) reported that adding 12 mg of manganese sulfate
100 kg of poultry meal afforded additional resistance to disease.
rot and Durand (1944) found that the Mn content of malignant and
nign tumors was much lower than that of healthy tissues. Sandstedt
t al. (1951) showed that daily supplementation with 0.5 g of manganese
11fate gave rapid recovery from acetonemia in cattle, and preventive
:eatment over the years greatly reduced the incidence of the disease.
lmofal (1961) found that the level of Mn in the diet was a decisive
.ctor in the occurrence of goiter. Kamchatrov (1959) and Hakimova at
w (1969) showed that excess dietary Mn affects thyroid metabolism.
ellis (1970) showed a depression of the protein-bound iodine fraction.
rlier Kamchatnov (1953) had noted a relationship between Mn content
feed items and the distribution of enzootic and non-enzootic goiter
gions. Angelica at al. (1965) and Antanova (1968) have reported that
supplementation significantly delayed the death of rats and increased
a survival rate after nitrogen mustard poisoning and coliform bac-
rial infection.
Weinberg (1964) discovered that certain bacteria had specific

[uirements for Mn, in excess of that needed for growth, in order to

 

 

 

 

 

 

 

21

oduce antibiotic, bacteriophage, and protective antigens. Antanova
968) and Antanova at al. (1968) reported that agglutinin response and
agocytic activity were greater with higher Mn intakes in rabbits when
unized with coliform and typhoid bacteria. Muraleedharan and Pande

968) found that with Mn-deficient diets, infection with Prosthogonimus

atus seemed to hasten death.
Mn Metabolism

1. Absorption

Little is known about the mechanism of Mn absorption from the
trointestinal tract. Scott at al. (1958) showed that a Mn compound
pable of dissolving and being converted to manganese chloride in the
id medium of the gastrointestinal tract can be absorbed.
The uptake of Mn by the intestinal mucosa is very rapid (Miller et
., 1972), and the element is bound to the serum beta—globulin fraction
all species studied (Panic and Ekman, 1967). While Saltman at al.
)56) thought that simple diffusion constituted the driving force for
a transport of Mn, Rothstein et a1. (1958) and Weed and Rothstein
’58) presented evidence for active transport for Mn. Gutowska et a1.
41) showed that the amount of Mn absorbed from a solution of manganese
fate was proportional to its concentration; an average of a third of
amount injected in the intestines was absorbed in two hours. There
no significant sex difference in absorption of Mn in chickens.
y reports have indicated that only 3 to 4 percent of an oral dose of
is absorbed (Britton and Cotzias, 1966; watson et al., 1973). But
1e et a1. (1971) and Brown and McCracken (1965) have reported sub-

tial Mn absorption. Pregnant sows absorbed up to 28 percent of

sted Mn (Gamble at al., 1971)-

 

 

 

 

 

 

 

22

Manganese absorption is affected by various factors present in
diet. Poll at al. (1967) and Hill and Holtkamp (1954) reported
t Mn was more readily absorbed at a lower than at a higher dietary
concentration. Cotzias and Greenough (1958) and Zajcev (1959) found
t Mn absorption was not affected by its valency state in the com-
:nds used. Many workers have reported that high dietary levels of
and P aggravated Mn deficiency due to reduction in_Mn absorption
Lderwood, 1971). Intestinal absorption of Mn was increased in rats
Le iron deficient (Pollack et al., 1965). Saltman et al. (1956)
1nd that Mn competitively inhibited Fe uptake and release by liver
.ces, which indicated that both Fe and Mn have a common pathway.
L0 and Edwards (1968) reported that Mn absorption was enhanced by
:thylenetriamine pentaacetic acid (DTPA) and decreased by ethylene-
mine tetraacetate (EDTA). Leibholz at al. (1962) found that pigs
casein diets containing 0.4 and 40.4 ppm Mn grew at a more rapid
e on less food per pound of gain than did pigs fed soybean protein
ions containing 11.8 and 51.8 ppm Mn. Davis et a2. (1962) found a
or in soybeans which tends to bind Mn and makes Mn unavailable.

Settle et a1. (1969) found no appreciable binding of Mn in feather

diets. Gilbert (1957) reported that thiamine, given in excess,
:ipitated a Mn deficiency, but Holtkamp at al. (1950) found no

ience of antagonism between dietary Mn and thiamine.

2. Excretion
Everson and Daniels (1934) observed that total Mn urinary
'etion is virtually constant, irrespective of age, and that fecal
xcretion varies directly with age and therefore with total dietary

ntake. ‘Many workers have reported that most of Mn administered

 

 

 

 

 

23
orally or intraperitoneally quickly appeared in bile and was excreted
in the feces, and very little was excreted in the urine (Kent and
McCance, 1941; Mahoney and Small, 1968; Starodubova, 1968). The pre—

dominance of the fecal route for Mn excretion has been verified in

 

simple stomached animals (Britton and Cotzias, 1966; Miller, 1973), in
sheep (Watson at aZ., 1973), in cattle (Miller et al.. 1973), and in
man (North et al., 1960; Cotzias and Greenbough, 1958).

Under normal conditions, the bile flow is the principal regulatory
mechanism of Mn excretion, and the concentration of Mn in bile can be

increased tenfold or more by the animal (Underwood, 1971). The other

 

routes of Mn excretion include pancreatic juice (Papavasiliou et al.,
1966; Burnett et aZ., 1952) and secretions of the duodenum, jejunum
and, to a smaller extent, the terminal ileum (Bertinchamps at al.,
1966). Excretion via the kidney is negligible normally or during
jaundice or after a marked oral dose. The administration of chelating
agents such as EDTA produces a marked rise in urinary excretion of Mn
(Maynard and Fink, 1956).

Lassiter at al. (1970) reported that the body pool is small and

 

:he body does not ordinarily accumulate Mn. Total body excretion is
:ontinuous and very nearly equal to intake, and much of the Mn in the
lody pool is replenished daily. Animals placed on a low Mn diet con—
:inue to excrete Mn, suggesting an obligatory loss of Mn (Zajcev, 1959;
tarodubova, 1968). The excretion of Mn administered parenterally was
uch lower on a 1.0 percent Ca diet than on a 0.6 percent Ca diet, but
aising P levels had no comparable effect on the excretion or retention
f Mn (Lassiter at al., 1970). Underwood (1971) noted that Ca influences

1 metabolism by affecting its absorption. The normal excretion of Mn

 

tom the body is prevented by rectal and biliary ligation (Papavasiliou

 

 

 

 

 

24
al., 1966), and there is no appreciable interdependence of these

tes of excretion (Bertinchamps at aZ., 1966).

3. Retention

 

Everson and Daniels (1934) reported that Mn retention varies
ersely with age of children and therefore intake. North et al.
60) showed that college women retained about a third of the absorbed
and pullets retained about the same amounts (Brown and MtCracken,
5). Gamble at al. (1971) showed that 7'days after the administra—

n of a radioactive dose of Mn, pregnant sows retained 26 percent of

 

oral and 78 percent of an intravenous dose. A highly significant
relation between intake and retention has been found (Mathers and

L, 1967; Murty, 1957), but Hughes at al. (1966) reported that only
nall change in tissue Mn level can be effected by a large change in
:ary Mn intake. There is a linear relationship between Mn turnover
level of dietary Mn intake, and the half-life of body Mn is

teased with increasing Mn intake (Britton and Cotzias, 1966). The
nrbed Mn is found principally in the liver and bone. At lower Mn
Lkes the liver retains more Mn than intestinal tissue, but at high
Lkes the latter retains more (Underwood, 1971). Passive transendo-
ial transport occurs immediately after intravenous administration
n, and about 70 percent of the blood Mn leaves the circulation each
te and is mainly taken up by the liver (Cotzias, 1958; Thomas,

). Borg and Cotzias (1958) showed that most of the endogenous Mn
ts in highly labile intracellular combinations.

Tissue Mn concentrations generally are remarkably constant even
gh consumption levels vary greatly (Underwood, 1971). These might

egulated through variable excretion rates (Britton and Cotzias,

 

 

 

 

 

 

25

6), and absorption differences (Howes and Dyer, 1971; Miller, 1973).
siter at al. (1970) showed that 0.9 percent dietary P caused signifi-
tly higher Mn retention of orally administered Mn than did 0.4 per—
t P, but there were no comparable effects on intraperitoneally
inistered Mn. Hughes and Cotzias (1961) found that administration
ogenous glucocorticoid hormone markedly affected the tissue distri—
on of radioactive Mn, but adrenalectomy did not affect retention
hes et al., 1966). Hill (1967) found that vitamin D reduced Mn
over, and Suso and Edwards (1968) reported that EDTA reduced the
ntion of intravenously injected Mn, and greatly increased Mn trans-
(Sahagian at al., 1967). Gamble et a1. (1971) reported that
gnancy in swine had no significant effect upon maternal tissue

antion, organ distribution or turnover rate of Mn. Fournier et al.

'2) reported increased Mn retention in all organs as a result of

:ose ingestion.
{Manganese Requirement

1. Factors Affecting Mn Requirement

There is a very wide margin of safety between the minimum and

c levels of Mn for all species. The minimum dietary requirements

depend upon the species, the criteria of adequacy employed, the

ical form in which the element is ingested, and the nature of the
of the diet (Underwood, 1971).

Manganese requirements differ between and within species. Chickens

re more Mn than any other species (Thomas, 1970). A level of 13

in the diet resulted in signs of perosis in White Leghorns and
ampshires but not in Rhode Island Reds (Pilla, 1958). Chondro-

ophy was confined to Barred Rock chicks only and did not affect

 

 

 

 

 

 

 

 

26

its Leghorns, and the response to Mn supplementation was greatest in
w Hampshires.and least in Leghorns (Golding at aZ., 1940). Gutowska
al. (1941) and Mathers and Hill (1967) reported no significant sex
fference in Mn utilization in chickens. But Barnes at al. (1941)
ported that female chicks were more sensitive to Mn deficiency in
e mother's diet than male chicks. And Pilla (1958) found that the
sponse to Mn was greater in males than females. There is evidence
owing that Mn levels necessary for growth are less for reproduction
Lojas et aZ., 1965; Grummer et al., 1950; Bentley et aZ., 1951a).
In attempting to define dietary requirements of Mn, the composi—
.on of the diet is crucial. There are interactions that occur in the
»0d or in the gastrointestinal lumen. Manganese interacts with Ca and
within the digestive tract. In 1939, Caskey and Norris observed that
iwas made unavailable by high levels of Ca and P in the diets of
ickens. High dietary levels of Ca and P have been shown to decrease
e growth rate and increase the incidence of perosis in chickens on
ets having Mn levels ordinarily adequate to prevent perosis, and
ditional Mn prevented the development of perosis (Underwood, 1971).
gus and Patton (1939) explained the perosis-producing action of
cium phosphate as being due, at least in large part, to the removal
Mn from solution in the intestinal tract by adsorption or chemical
bination. High levels of ferrous citrate also increased the inci~
ce of perosis, presumably through similar intraluminal action (Wilgus
Patton, 1939). Addition of ferric oxide to a Mn and Cu mixture
ed to offset its beneficial effect on hatchability (Hoodengoorn,
0). In intracellular fractions, the concentration of Mn and Fe have
n shown to have a reciprocal relationship (Thiers and Vallee, 1957).

re is some evidence of dietary Mg-Mn and Zn—Mn interactions

 

 

 

 

 

 

 

 

 

27

kemore et al., 1937; Cotzias, 1960; Sahagian at aZ., 1966). Diez—

d et a1. (1968) and Kolomijceva and Veznesenskaja (1968) have

rted interrelationships between Fe, Cu and Mn metabolism.

In some other circumstances, high levels of dietary thiamine intake
ease body Mn storage (Hill and Mathers, 1954), but Sandberg et a1.
9) found that the state of thiamine deficiency caused great increases
in retention. Anderson and Parker (1955) found no thiamine level
act on liver and heart Mn content. Holtkamp et a1. (1950) found no
lence of antagonism between thiamine and Mn. Perla and Sandberg
39) reported that high thiamine levels causing low reproduction rates
be counteracted by increasing Mn intakes. Riboflavin, at high levels,

shown to complicate perosis in ducklings (Turton, 1953).

 

The protein source of the diet may affect Mn metabolism. The

 

Llability of Mn to chicks was better when either fishmeal or dried

1 milk was the protein source as compared to soybean meal (Morimoto
rZ., 1959; Kealy and Sullivan, 1966). Davis et a1. (1962) showed

: soybean protein contains a component which combines with Mn leading
:onditioned deficiency. Settle et a1. (1969) found no such binding
In when feathermeal was fed as the source of protein.

The Mn compound fed may determine Mn availability and requirements.
.emer at al. (1940) showed that precipitated MnCO3 protected against
sis at a lower level than the naturally occurring carbonates which
ed useless even when used in large amounts due to low solubility.
ible and Bandemer (1942) reported that chemical forms of Mn com-
ds as diverse as carbonates, oxides, sulfates and chlorides were
11y valuable as sources of Mn in poultry rations. Anke et al.

7) and Watson et a2. (1970) have shown that the chloride and sulfate

etter utilized than the oxides in cattle. Lyons (1939) and

 

 

 

 

28

eshi et al. (1963) found that Mn supplied in rice bran appeared to

3 well utilized as the inorganic sulfate. The method of administra-
l of Mn is important in determining its effectiveness. Caskey and
'is (1939) showed that small amounts of Mn injected intraperitoneally
: more effective than ingested materials against perosis.

A number of feed additives have been shown to either depress or
.nce Mn availability to the animal. The inclusion of chlortetra-
.ine in low Mn diets for chickens reduced the incidence of perosis
he pullets but not in their progeny (Pepper at al., 1952, 1953).

on (1955) showed that giving estradiol increased plasma and liver

 

 

| Hart (1953, 1954) reported that giving Vevoron, an antithyroid
aration containing methylthiouracil for fattening, significantly
essed the Mn content of the liver. Hydrazine administration can
e symptoms in animals similar to those of Mn deficiency (Thomas,
).

There are other factors which may influence Mn requirements.
stiansen at al. (1939) concluded that sunlight exerts a sparing
on on the hens' Mn requirements. Urban (1959) showed that hepa-

omy aggravated Mn deficiency.

2. Mn Requirement of Swine

There are conflicting data in the literature on the exact Mn
irements of pigs for proper growth, skeletal development and repro-
Lon. Keith et al. (1942), using a high corn ration fortified with
rals and containing 11 to 14 ppm Mn, found that the growth of pigs
1ot impaired but skeletal development was poor, and a supplemental
L of 50 ppm Mn prevented skeletal deformities but did not cure them.

3r et al. (1940) had a similar response with 60 ppm supplementation.

 

 

29

hnson (1940) reported that 6 ppm was sufficient for successful repro-
ction of sows; and satisfactory growth was obtained on 0.3 ppm Mn,
t reproduction was unsatisfactory and, at this level, tissue Mn
ntent was significantly depressed. Johnson (1944) showed that pigs
ew well from weanling to market weight on rations containing 7 to 10
m Mn. Grummer et al. (1950) reported that a diet containing 12 ppm
was adequate for skeletal development.and growth but not adequate
r optimum reproductive performance. When the same diet was supple—
nted with 40, 80 and 160 ppm Mn, the highest average daily gains were
tained on the 40 ppm level. Higher supplementation had no added
nefit. Plumlee et a2. (1956) reported that dietary Mn concentrations
inging from 0.5 to 34 ppm Mn did not show significant differences in
Lg performance. Speer et al. (1952) showed that pigs from sows
[pplemented with 70 to 90 ppm Mn performed best during growing and
Lttening as compared to those from unsupplemented groups. Leibholz
: a2. (1962) defined the baby pig Mn requirement at 0.4 ppm Mn for
ximal growth rate. Leibholz et a1. (1962) and Newland et al. (1961)
lund independently that sows farrowed or produced normal fetuses when
d 89 to 117 ppm Mn or 6 to 100 ppm Mn in their diets, respectively.
e Mn requirements for growth of pigs are extremely low, well below
e levels ordinarily found in practical swine diets, although the

tional Research Council (NRC, 1973) recommended 20 ppm Mn in the diet.

3. Mn Requirement of Other Species
The Mn requirements of many species have been estimated by
my investigators and reviewed by Thomas (1970) and Underwood (1971).
ds have higher Mn requirements than mammals. The best evidence

icates that to prevent deformities in the fetus, cows should receive

 

 

 

 

 

 

 

30
diet containing 20 ppm Mn. In calves 8.6 mg per day were not suffi-
ient but 36 mg per day were optimum for growth. Poultry requirements
ave been put at 40 to 70 ppm Mn in the diet. Children require at
east 1.2 mg per day; and older humans 3 to 5 mg. Requirements for the
at have been set at 0.5 to 1.0 mg per day, and the low amount of 1 ppm
1 the diet produces fetal abnormalities. Rabbits require about 1 mg

3r day, and sheep 50 to 60 ppm on a feed dry matter basis.

Mn Toxicity,

 

Mn is one of the least toxic of the trace elements to mammals and
ers. Richards (1930) fed pigs 3.5 g of manganese citrate daily for

.ne months without any adverse effects. Mussehl and Ackerson (1939)

 

lOWEd that turkeys can tolerate 385 ppm Mn in their diets and Insko
: a2. (1938) reported that hens tolerated 600 to 1000 ppm Mn. However,
.hers have reported adverse effects due to high level feeding of Mn.
mimura (1938) found that rabbits fed 0.5 to 6 g per kg bodyweight
.ily were stunted and their bone development impeded. Similar effects
ve been shown in rats and cattle (Chornock et a2., 1942; Cunningham
aZ., 1966). Wessinger et a1. (1943) found that injections of solu—
ons containing 180 to 975 ppm Mn as MnCl2 into white rats caused
mel hypoplasia, interrupting amelogenesis in the apical quarter of
zone of matrix formation. Heller and Penquite (1937), feeding a
ion containing 4800 ppm Mn, showed the element to be highly toxic
young chickens.
High level feeding of Mn interferes with Fe, Cu and P metabolism
derwood, 1971). Urinary Cu excretion is depressed and Cu retention
the tissues is increased, causing a microcytic, hypochromic anemia in

3 fed very high Mn levels (Gubler et al., 1954). Earlier, Edgar

 

 

 

 

 

 

31

942) supplemented sheep rations with 25 mg of Mn as the sulfate and
mg of Cu as the sulfate daily and showed large but non-significant
creases in liver Cu levels over those of the controls receiving copper
[fate alone. Experimentally, dietary Mn levels causing a suppression
hemoglobin formation are 1000 to 2000 ppm for anemic lambs (Hartman
aZ., 1955), 5000 ppm for normal lambs (Robinson et al.: 1960), 1250
n for mature rabbits (Matrone et al.. 1959), and 2000 ppm for baby
gs (Matrone et aZ.. 1959). Hartman et al. (1955) and Cunningham at

(1966) reported that high levels of dietary Mn reSulted in decreased
acentrations of Fe in liver, kidney and spleen of ruminants, and
pressed hemoglobin formation.

Blakemore et al. (1937) found that the Mn content of pastures in
stricts where lactation tetany was prevalent was 700 ppm on a dry
tter basis as compared to 50 ppm of pastures on which tetany had never
an recorded. The feeding of Mn to rabbits, sheep and cows in amounts
pplied in the pastures on which tetany occurred brought about transi-
y falls in levels of Mg in blood. Robinson et a1. (1960) and
ningham at al. (1966) found that cattle fed high Mn levels produced
3 rumen propionic acid. Keith et a1. (1942) showed that growing
9 fed on diets containing 2000 ppm Mn grew poorly, lost weight and
etite. They vomited and had nausea, diarrhea and dermatitis.
mmer et a1. (1950) showed that pigs do not tolerate high levels of
tary Mn since 500 ppm in the diet reduced growth rate, feed
iciency and depressed appetite of growing and finishing swine.

Leibholz at al. (1962) showed no toxicity signs in baby pigs fed
0 ppm Mn; however, there was evidence of reduced growth rate at
0 ppm. This work showed a high tolerance by the baby pig and a

siderable margin of safety between levels of Mn.likely to be

 

 

 

 

 

 

 

 

32
ngested in the diet and detrimental levels. In man the contamination

f air and water by large amounts of Mn causes locula manganica, dis-

 

urbance of the extrapyramidal tract and atrophy and disappearance of
Lerve cells of the globus pallidus (Cotzias, 1958; Belani et al., 1967).
:otzias (1968) and Mena et a2. (1968) showed a decreased turnover of

m in patients suffering from chronic Mn poisoning.

 

 

 

EXPERIMENTAL PROCEDURES

Introduction

 

Four experiments involving 104 pigs were conducted to study Mn
:abolism in swine. These were:

Experiment 1. The relative availability of Mn from a 16% corn—soy

 

3al diet and the basal diet supplemented with 10 ppm of Mn from

soaouzo, Mnco3

Experiment 2. Study of the gastrointestinal flux pattern of Mn

or MnO for the growing pig.

 

 

om different Mn sources using chromic oxide (Cr203) as an indicator.

 

Experiment 3. The effect of high level Ca and P supplementation

 

i an inverse Ca—P ratio on Mn utilization by the growing pig.

Experiment 4. The Mn requirement of the baby pig from sows fed a

 

Mn diet.
Yorkshire, Hampshire and Yorkshire—Hampshire crossbred pigs from
Michigan State University herd were used. All trials were conducted

the university swine farm facilities.

Experiments

1. Experiment 1

 

Before the diets for this experiment were made, three Mn come

nds (manganous sulfate, manganous carbonate and manganous oxide) were

jected to a 0.4% HCl availability study.

 

 

 

 

 

34

A liter of 0.4% HCl was heated in a 1500 ml beaker to a constant
mperature (37°C) in a gyrotory water bath shaker, MOdel G76.l One
am of each compound, ground to pass through a 100 mesh screen, was
ded; the temperature and agitation were maintained for one hour. The
sulting solution was filtered at once through a dry Whatman #42 filter
per, discarding approximately the first 50 ml. The filtrate was
oroughly shaken, sub-sampled into three 20 ml aliquots, and Mn was
termined by atomic absorption spectroscopy.

Twelve crossbred weanling pigs, 9 males and 3 females, from two

 

tters, were allotted to the four diets shown in Table l, equalizing

r sex, litter and weight. The supplemented diets were made using the
ne compounds used in the above study. The basal diet was supplemented
:h 10 ppm of Mn from MnSO

0, MnCO or MnO to make diets 2, 3, and

4'H2 3
respectively.

The pigs weighed about 8 to 10 kg initially. They were housed in
lividual stainless steel metabolism cages for a 7-day adjustment
:iod followed by two balance trials. The first was a replicated
:in square design in which all animals were fed all the diets during
1r collection periods. The second was a split-plot design in which
:ee repeated collections were made on the animals maintained on the
1e diets. The pigs were removed from the cages three times daily and
lividually fed an amount of food and water which could be consumed
:hin a 5— to lO-minute period. The feed was mixed with water to make

:lurry for quick consumption. Following feeding, the pigs' mouths

'e wiped clean to avoid contamination of excreta. The pigs were then

:urned to the cages.

1New Brunswick Scientific, New Brunswick, N.J.

 

 

 

 

35

 

 

 

 

 

 

 

3LE l. DIETS USED IN EXPERIMENT 1
Diet

gredient Basal + Mn804'H20 + MnCO + MnO
tn, shelled, ground 79.2 78.2 78.2 78.2
ybean meal, dehulled 17.9 17.9 17.9 17.9
solvent (49% CP)
lt 0.5 0.5 0.5 0.5
nestone, grd (38% Ca) 0.9 0.9 0.9 0.9
calcium phosphate 1.0 1.0 1.0 1.0
M premix1 0.5 0.5 0.5 0.5
SO4'H20 premix 1.0
CO3 premix 1.0
0 premix 1.0

100 100 100 100

l

vel of CP (calculated), % 16 16 16 16
vel of Mn (analyzed), ppm 16.2 26.0 24.8 27.0

 

1See Appendix A, Table A-1.

 

 

 

 

5— r_J.-r-- .,51

36
Fecal and urine collections were made over a 3-day period, with
-day intervals in between each collection. Feces were separated from
’ine by means of a fine screen placed over the urine collection funnel.
e pigs consumed near ad Zibitum quantities of feed daily, and constant
ily feed intakes were maintained throughout the balance periods.
consumed feed was collected after each feeding during the 3-day

llection period to get an accurate measure of feed intake during the

lance trial.

Feces were oven-dried for 24 hours, equilibrated to room temperature

 

r 12 hours, weighed, ground and stored in sealed plastic containers.
fused feed was air-dried, weighed and discarded. Urine was collected
polyethylene containers and acidified with 6N HCl. Following the
llection period, the urine volume was recorded and 100 ml aliquots
re stored in acid-washed polyethylene bottles at 4°C.

The animals were bled weekly from the anterior vena cava before
a start of each collection period for the determination of serum
(aline phosphatase and serum Mn concentration. All the animals were
[led after the second balance trial. The following tissues were
Llected, weighed and stored in a freezer in polyethylene bags: liver,
lneys, spleen, testes, heart, left femur, 8th left rib, pancreas and
a muscle from the left leg. Hair samples were collected from the
Lmals at the beginning and at the end of the experiment from the loin

gion of each animal.

. Experiment 2

 

The gastrointestinal flux pattern of Mn from different Mn sources.
'omic oxide was added (at 0.3%) to each of the diets used in Experiment

Pigs were fed these diets for a period of 4 days before they were

 

 

 

 

37

Feces voided were weighed and grab samples of the feces were
for further analyses.
.gs were killed one and one—half to three hours postfeeding. The
:ary tract was quickly exposed and sectioned into the stomach,
. small intestine, caudal small intestine, cecum, colon and

The sections were ligated to prevent movement of the contents.
. were removed quantitatively from each section, weighed, and the
:rmined on a small portion. The remainder was thoroughly mixed
'ided into 20 g portions. These were placed in polyethylene bags
zen for later analysis.
.e indicator method for determining digestibility was used to

e absorption and secretion of Mn along the gastrointestinal

The following equation was used:

. . . _ % indicator in feed % nutrient in feces
tlblllty — 100 - (% indicator in feces % nutrient in feed ) 100

on and Harris, 1969). In order to measure the net absorption
retion in the different sections of the alimentary tract as the
moved posteriorly, two consecutive sections of the tract were
relation to the above equation. The digesta in a given section
sidered to be the feed for the next posterior section. The

in the second section would be equivalent to the feces for the
tion using the ratio technique equation. Thus net absorption or
on was calculated in the following alimentary sections as
ed:

Stomach - using feed as feed and stomach digesta as feces.

Cranial small intestine - using stomach digesta as feed and

small intestine digesta as feces.

 

 

 

 

 

 

 

38

3. Caudal small intestine - using cranial small intestine digesta
ed and caudal small intestine digesta as feces.

4. Cecum - using caudal small intestine digesta as feed and cecal
ta as feces.

5. Colon - using cecal digesta as feed and digesta in the colon
ces.

6. Rectum - using colon digesta as feed and the feces voided as
5.
With this method, a positive value indicated net absorption from
section and a negative value, net secretion into that section of

gastrointestinal tract.

Experiment 3

 

The effect of high levels of Ca and P supplementation and an inverse
ratio on Mn utilization by the growing pig.

A feeding trial using a 23 factorial design was conducted. Eighty

Ling pigs weighing approximately 8 kg were randomly allotted to

aatments shown in Table 2, equalizing for sex, litter and weight.

1g the trial, the animals were housed in confinement on slotted

rete floors. Feed and water were provided ad Zibitum. The pigs
weighed every 2 weeks and 2 animals per lot, selected at random,
bled every 4 weeks to monitor changes in blood constituents.

1 samples were taken from the anterior vena cava.

After 10 weeks, the experiment was terminated when the animals

led an average of 60 kg. Four animals from each lot, including the

.mals that were regularly bled, were slaughtered at the university
Laboratory and tissue samples collected for chemical, physical

istopathological analyses. The tissues collected included bones,

 

 

 

 

 

 

E 2. DIETS USED IN FEEDING TRIAL (EXPERIMENT 3)

 

% (calculated)
(calculated)

ppm (analyzed)

1

.7

1.4

.7

1.4

.35

.35

15.3 56.4 15.9 55.7 15.2 56.1 16.0 55.8

r‘

 

 

 

 

 

 

753 743 789 779 791 781 761 751
ean meal de-
llled solvent, 188 188 183 183 181 181 186 186
+9% CP)
5 5 5 5 5 5 5 5
:stone 17 17 23 23
Llcium phosphate 26 26 12 12 19 19
premixl 5 5 5 5 5 5 5 5
iremix 10 10 10 10
biotic3 1 1 l 1 l l l 1
sodium phosphate 22 22 5 5
1000 1000 1000 1000 1000 1000 1000 1000
1MSU vitamin - trace mineral premix without manganese. See

ndix A, Table A-1.

2Containing 4000 ppm Mn from Mnsoa-

H2

3Containing 22 g of chlortetracycline per kg.

 

O reagent grade.

 

 

 

 

 

t, pancreas, liver and the left kidney. The bones taken included
first left rib and the lateral and medial metacarpals from the

leg. The medial metacarpals were split longitudinally, fixed in
ered 10% formalin, sectioned and stained with hematoxylin and eosin
histopathologic examination. Two sections were taken from each

, one through the epiphyseal cartilagenous plate from the proximal
of the bone and one section through the diaphysis. Sections of the
hysis and diaphysis were coded as follows: 1, normal, if they had
little cartilage and osteoid; 2, very slight change, cartilage and
oid persisting in the bony spicules distal to the epiphyseal
ilagenous plate or distal from the periosteum of the diaphysis;
3, slight change, 4, moderate change, or 5, severe change, depending

the degree of persistence of cartilage in bony spicules.

Experiment 4

 

The Mn requirement of the baby pig from sows fed a low Mn diet.
it one—month pregnant first litter gilts were fed a low Mn diet
an in Table 3 throughout the remainder of the gestation period.
3r farrowing, 20 baby pigs were taken from the sows at 5 days of age
placed in stainless steel rearing cages equipped with stainless
21 feeders and water troughs. The room temperature was maintained
30°C for the period of the trial. The pigs were weaned to the basal
purified diet (no. 1) shown in Table 4. Pigs were taught to consume
feed by placing small amounts in the animal's mouth. Some pigs
lily adapted to the dry feed, but many were affected with diarrhea,
lme weak, refused to eat and eventually died.

After adapting to the diets, 12 healthy pigs were randomly allotted

:he three dietary treatments shown in Table 4. The pigs were fed

 

 

 

 

 

 

41

3. LOW MANGANESE GESTATION DIET (EXPERIMENT 4)

 

 

Ingredient Amount, kg2

Corn 1915

Limestone 20

Dicalcium phosphate 30

Salt 10

. l

VTM premix 10

Vit. E premix 5

Lysine (50%) 10
2000

1See Appendix A, Table A-1 supplied all the trace minerals
: Mn.

2Level of manganese 11.2 ppm as analyzed.

 

 

 

 

 

 

42

LE 4. PURIFIED DIETS USED IN EXPERIMENT 4

 

 

 

 

Diets
n l 2 3
in 30 30 30
lose 55 52 46
ulose 3 3 3
d 5 5 5
. . 1

amin premix 1 l 1
premix2 3 9
eral premix3 6 6 6

100 100 100
concentration, ppm 0.46 2.67 6.34
analyzed)

 

r_.

1See Appendix A, Table A—3.

2
Manganese premix containing 100 ppm Mn from MnSOa’HZO, J. T.
at reagent grade.

3See Appendix A, Table A-1.

 

 

 

 

 

 

43

Zibitum and had free access to water, which was changed 3 times a
. All feed was weighed daily and individual feed consumption was
orded. Pigs were individually weighed every 4 days throughout the
day trial. One pig (234-4F) on the basal diet died suddenly and the
se of death was determined to be heart failure. The pigs were bled
the 9th, let, and 28th day of the feeding trial.

After the feeding trial, the pigs were used for a Mn balance
iy. Another pig (241-11) on diet 3 was eliminated from the study

to an infection in the left rear leg. The pigs were housed, treated,
fed as described in Experiment 1. The collection and treatment of
feces, urine and refused feed was similar to the procedures described

Experiment 1.
Analyses
l. Hematological Determinations

a. Hemoglobin. In Experiments 1 and 3, hemoglobin was

 

armined by the cyanmethemoglobin method of Crosby at al. (1954).

aleman Junior II spectrophotometer was used to determine optical

sity at 540 nm.

b. Hematocrit. Hematocrit was determined by the micro method

 

Sovern at al., 1955) in Experiments 1 and 3. Blood samples were

trifuged for 5 minutes at 10,000 rpm in an International "Hemacrit"

trifuge.

2. Physical Determinations

a. Bone. In Experiment 1, the left femur and the 8th left rib

3 removed and cleaned of all connective tissue and periosteum and

 

 

 

 

 

 

44
d in air—tight polyethylene bagS. The ribs were used for specific
ty determinations and the femurs for breaking strength and related
eters.
In Experiment 3, the 5th metacarpal was removed from the left foot,
ed of connective tissue and stored in a cold room in air—tight
thylene bags. The first left rib was obtained and treated as the
arpal. Bone strength and density tests were made on both the
and the metacarpals.

Specific gravity was determined according to the following formula:

= weight in air
weight in air - weight in water

 

Specific gravity

In Experiments 1 and 3, the strength of the metacarpals, femurs,
ibs was determined using an Instron Testing Instrument, Model—TT
equipped with an FM—compression load cell having 100 kg full scale.
toss—head speed and chart speed were 0.2 cm/minute and 2 cm/minute,
:tively. Metal fulcra were used to support the metacarpals and

and the distance between fulcra was maintained constant at 3 and
respectively. The femurs varied in size and therefore the dis—
between supporting fulcra varied from bone to bone. The formulas
alculating the various strength characteristics were those

ibed by Miller et al. (1962) and are as follows:

iaximal bending moment M = Wl/4
3 3
doment of inertia I = (Bd - bd )/64
iaximal stress S = MD/ZI
E = Wl3/48Iy

Elasticity

lInstron Engineering Corporation, Canton, Mass.

 

 

 

 

 

45

W = maximal load

1 = distance between fulcra
B,b = outer and inner horizontal diameter
D,d = outer and inner vertical diameter

y = deflection at center of bone when load W is applied

3. Chemical Determinations

a. Blggg, Upon withdrawal, the blood was placed in acid-
3d centrifuge tubes, allowed to clot and centrifuged at 2000 g for
Lnutes. The cell-free serum was then harvested and placed in acid-
ad vials. Small amounts were stored in the cold room (4°C) for
:mination of alkaline phosphatase activity, and the remaining serum

Les were frozen for subsequent mineral analysis.

(1) Serum alkaline phosphatase. Determination of serum

 

.ine phosphatase activity was made according to the procedure
ribed in Sigma Technical Bulletin No. 194 (1963). The Sigma 104
thatase substrate was used in the enzyme activity determination.
:kman Model DU spectrophotometer was used for optical density

'minations in Experiments 1, 3 and 4.

(2) Serum calcium and phosphorus. To one milliliter of
I from Experiment 3 was added 4 m1 of 12.5% TCA. Serum proteins
.pitated by the 12.5% TCA were centrifuged out at 2000 g for 15
.es, and the resulting protein—free supernatant was diluted 1:1 with

.tium mixture A1 to suppress phosphate interference. Calcium was

l._.k

1
).

o o 0d
61.0 g strontium chloride (SrCl2 6H20) + 10.0 g sodium chlori e

 

 

 

 

 

 

 

46
determined at 422.6 nm by atomic absorption spectrophotometry using
Jarrell-Ash1 Model 82-516 spectrophotometer equipped with a Hetco
1 consumption burner and an air-hydrogen flame, as described by
ey et al. (1967).
For phosphorus determinations the Gomorri (1942) method was used.
optical density was determined on a Coleman Junior 11 spectropho-

ter at 700 nm after a 45-minute incubation period.

(3) Serum magnesium. Cell-free serum samples from Experi-

 

s 3 and 4 were diluted 1:100 with strontium mixture B2 to avoid
phate interference. Magnesium was determined with a Jarrell—Ash
ic absorption spectrophotometer at 285.2 nm in Experiment 3, and
xperiment 4 an Instrumentation Laboratories, Inc.,3 Model 453

ic absorption spectrophotometer was used.
(4) Serum mapganese—neutron activation

(a) Principle. Mn in serum from Experiments 1 and

 

3 determined by modification of the neutron activation procedure of
at al. (1968). The principle involved exposure of a small amount

erum to a thermal neutron flux in a nuclear reactor and the follow-

nuclear reaction occurred:

55Mn(n, y)+56Mn

lJarrell-Ash Co., Waltham, Mass.
2
L).

3Instrumentation Laboratories, Inc., Lexington, Mass.

30.5 g strontium chloride (SrClz-6H20) + 5.0 g sodium chloride

 

 

 

 

 

 

47
56
The induced Mn has a 2.56 hour half-life and emits gamma rays of
. . 5
5 Mev. The act1v1ty of 6Mn can be measured by using gamma-ray
ctrometry, comparing the areas of the 0.85 Mev gamma-ray photopeaks

h those of the induced standards (Anong-Nilubol et al., 1968).

(b) Sample preparation. One milliliter of cell-free

 

um was pipetted into an acid-washed 250 ml Phillips beaker and 5 ml
concentrated HNO3 acid were added. The contents were heated gently
boiling and evaporated to dryness. Three milliliters of 1N HNO3
e added and heated again gently to boiling. After cooling, the

ume was made up to 5 ml. A like method was used to prepare the

ndards and nitric acid blanks.

(c) Irradiation. Four milliliters were pipetted into

 

yethylene vials,1 1.5 cm in diameter. The vials were thoroughly
aned (Jacobson at al., 1961)2 before loading. The vials were heat—
led, loaded into and activated by the MSU Triga Mark 1 reactor.3
samples were subjected to a thermal neutron flux of 2 X lOlzn/cmz/
for a period of 15 minutes. The samples were removed from the

ctor and the radioactivity of the polyethylene vials and samples was

the mrem/hour range and presented no special handling problem.

(d) Radiochemical separation. Radiochemical separa—

n was performed according to the method of Hahn at al. (1968). The

adiated samples were transferred to separatory funnels. The Vials

11.5 cm diameter — Olympia Plastics, Los Angeles, Calif.

2See Appendix A, Table A—4.

3Gulf General Atomic, Inc., San Diego, Calif.

 

 

 

 

 

48
rere rinsed with 8 m1 of deionized, distilled water. Two drops of
!.l% brilliant yellow were added and the pH was adjusted to neutral
Lsing 5N NH4OH. Then 2 ml of 5.7% sodium diethyldithiocarbamate and
. m1 of carbon tetrachloride were added. The contents were shaken for
:xactly 3 minutes and 4 ml of the organic layer were pipetted into

:lean vials for counting.

(e) Counting procedure. Counting was done about one

 

[our after the samples had been removed from the reactor on a 5.2 multi-
.hanne1 analyzer - computer series one-thirty1 utilizing a 3" X 3" NaI
rell crystal.2 The voltage was set at 1100 volts. The counts of the
.ntegrated areas under the peaks were corrected for both background,
Lecay time and blank. Sample counts were compared to standard counts

’or quantitation.

(5) Serum manganese - flameless atomic absopption. Mn in
.he serum samples from Experiment 4 was determined by flameless atomic
bsorption spectrophotometry on the Instrumentation Laboratories, Inc.,
bdel 355 accessory to the Model 453. Serum samples were diluted 20%
7v. The diluted serum was divided into three 25 ul aliquots. To all
Tut one of these were added known amounts of manganese, 10 ul of either
~or 10 ngm Mn/ml. The solutions were then placed on the tantalum
'ibbon and analyzed at 279.4 nm.3 The samples were dried, pyrolyzed for

'0 to 120 seconds and analyzed at a higher temperature than the one used

.uring pyrolysis. The Mn in the serum was calculated using the method

f additions (Slavin, 1968).

.—‘

1Tektronic, Inc., Portland, Ore.

2Packard Model, 1212 WSP serial 101-769.

3See Appendix A, Table A—5.

 

 

 

 

 

 

49
b. Bone. The cleaned ribs and metacarpals from Experiments
nd 3 used in the strength and density studies were used in the

mical analyses as well.

(1) Bone ash. The bones were cut in small pieces with a
er hand saw, wrapped in cheesecloth and extracted 24 hours with
olute ethanol and 24 hours with anhydrous diethyl ether in a Soxhlet
ractor to remove water and fat. The dry, fat—free bone was ashed in
uffle furnace at 600°C for 18 hours. The percent ash was calculated
m the following formula:

weight of ashed bone X 100 = a d f t-f b .
weight of dry, fat-free bone A aSh on a ry, a ree a51s

(2) Bone magnesium, manganese, calcium and phosphorus. The

ed bone was finely ground and approximately 300 mg of the powdered
e ash were dissolved in 5 ml of 6N HCl. Two milliliter aliquots of
resulting ash solution were diluted 1:1 with strontium mixture A and
was determined on a Jarrell—Ash atomic absorption spectrophotometer at
.4 nm. Bone Mn was expressed as ppm on a dry, fat—free basis.

The remainder of the ash solution was diluted 1:20 with deionized,
tilled water for further mineral analysis. The resulting solutions
e further diluted 1:100 with strontium mixture B and Ca and Mg were
ermined by atomic absorption spectrophotometry as previously described
blood serum. Phosphorus was determined by the colorimetric method
viously described for serum inorganic P. Bone Ca, P and Mg were

ressed as percent of the dry, fat-free bone.

 

 

 

 

 

 

50

c. Feedz feces and digesta

(l) Manganese. A wet ashing procedure was used. A 0.5
o 1.0 g sample was weighed into an acid—washed 250 ml Phillips beaker.
me milliliter of strontium mixture A was added and the contents

igested in 60 m1 of concentrated HNO acid on a hot plate to near dry—

3
ess and cooled. A second digestion with 7 ml of 72% perchloric acid
as performed. The contents were protected from excessively rapid
vaporation by a small water glass. They were heated to near dryness,
ooled and samples diluted to volume with deionized, distilled water.
tandards were prepared in a like manner. Mn content was determined
y atomic absorption spectroscopy using a Jarrell-Ash Model 82-516
pectrophotometer for feed and fecal samples from Experiments 1 and 3.
br digesta, feed and fecal samples from Experiments 2 and 4, Mn was

etermined with the Instrumentation Laboratories, Inc., Model 453 atomic

bsorption spectrophotometer.

(2) Chromium. Chromium was determined by the method of
olin et a1. (1952). A 100 to 500 mg sample of feed, feces, or digesta
. 1
as weighed into a 50 ml Erlenmeyer flask and 5 ml of oxid121ng reagent
ere added. The flask was then heated on a hot plate to digest the
dxture until it was clear. The mixture was cooled and 2 m1 of 72%
erchloric acid added and reheated. The flask was cooled to room
emperature and diluted to 50 ml using distilled, deionized water. The
amples were then read at 470 nm using distilled, deionized water as a

lank. The standard curve was prepared by oxidizing known amounts Of

he reference chromic oxide and diluting as described above.

 

+ 150 ml distilled deionized water +

l
' m m 1 bdate
10.0 g SOdlU. O y d and 200 ml 72% Perchloric acid-

50 m1 concentrated sulfuric aci

 

 

 

 

 

51
d. HEEES' A twofold concentration and digestion procedure

devised due to the low Mn content in urine. Twenty milliliters of

me were pipetted into a Phillips beaker, 1 ml of strontium mixture

as added and the contents were digested with 50 ml of concentrated

3 acid to near dryness. The flasks were cooled and diluted to 10 ml
h deionized, distilled water. Manganese was determined by atomic
orption spectroscopy on the Jarrell-Ash unit for urine from Experiment
nd on the Instrumentation Laboratories, Inc., Model 453 atomic
orption spectrophotometer for urine from Experiment 4. The method

specifications of determination were as described previously.

e. Tissues. The tissues included liver, kidney, pancreas,
rt, spleen, testes and muscle from both Experiments 1 and 3. Mag—
ium and P were determined on only the liver, kidney, pancreas and
rt samples from Experiment 3, and Mn was determined on all the

ples from both experiments.

(1) Tissue dry matter. Approximately 2 g samples were
ghed into disposable aluminum dishes and dried in a vacuum oven for

iours at 90°C. Dry matter was calculated as follows:

tissue dry weight X 100

Percent dry matter = tissue fresh weight

(2) Minerals. Tissue homogenates containing about 1 to
0f the tissues were pipetted into 250 ml Phillips beakers and a wet
Lng procedure was used as described previously for feed. The digesta
3 appropriately diluted and Mn, Ca and Mg were determined by atomic

’rPtion spectroscopy and P by the colorimetric method described

riously.

 

 

 

 

 

52
f. figig, Hair samples were soaked for 15 to 20 minutes in
aionized, distilled water, drained on filter paper and then placed
n 95% ethanol for 15 to 20 minutes to remove adhering contaminant
aterials. After removing from the ethanol, the samples were air—
ried and weighed into a 250 m1 Phillips beaker and wet-ashed. Mn was

.etermined by atomic absorption spectrometry as reported previously.

). Statistical Analysis

 

All data from Experiments 1, 2, 3, and 4 were subjected to analysis
3f variance on a CDC1 3600 computer at the Michigan State University
Computer Laboratory Center. The same computer was used to calculate
simple correlations. Differences between means were determined by

Tukey's test for non—additivity.

———.___.______

1Control Data Corporation, Minneapolis, Minn.

 

 

 

 

 

 

 

RESULTS AND DISCUSSION

Experiment 1: The relative availability of Mn from 16% corn-soy

basal diet supplemented with 10 ppm of Mn from either MnSO4°H20,

M'nCO3 or MnO for the growing pig

1. In vitro Solubility of Manganese Compounds

The solubility of manganous sulfate monohydrate (MnSO4-H20),

nanganous carbonate (MnCO3) and manganous oxide (MnO) is shown in
Table 5. The sulfate was soluble in water but the carbonate and oxide
rere practically insoluble. The sulfate and the carbonate were equally

soluble in 0.4% H01 but the oxide was slightly less soluble. These

findings are in agreement with those of Watson at al. (1971).

TABLE 5. CHEMICAL COMPOSITION AND SOLUBILITY OF DIFFERENT MANGANESE
COMPOUNDSl

 

Water 0.4% H01
Mn pH _pH
iompound % % Solubility Initial Final % Solubility Initial Final

nSO4°HZO 32.6 96.3 7.0 4.15 100.0 1.03 1.06
11003 44.3 0.91 7.0 7.70 100.0 1.03 1.04
no 65.0 0.12 7.0 7.65 89.5 1.03 1.10

 

1Water-bath was maintained at 37°C.

The pH dropped sharply after the sulfate dissolved in water (Table
, presumably due to dissociation of manganous sulfate and the formation

53

———_7

 

 

 

 

 

54
a predominantly acid medium. With the carbonate and oxide, the pH
e slightly. The pH changes after these compounds were dissolved in

% HCl were very small.

2. Mn Availability Studies Using a Split-Plot Design (where
Lmals were fed their respective experimental diets for three consecu-
ve Mn balance trials and three blood collections)
Weight gains, physical bone measures, and tissue Mn concentra-
ons were not affected significantly by source of Mn (Tables 6 and 7).'

imals receiving supplemental MnCO had significantly (P<0.05) heavier

3
.vers as a percent of bodyweight than those of animals receiving sup-
.emental MnSOa'HZO. The kidneys, hearts and testes as a percent of
Idy weight of animals on the basal diet were slightly heavier than
rose of animals on the other diets (Table 8).
The physical bone measures presented in Table 9 did not show any
vnsistent pattern of variation with respect to Mn sources. Mn from
.1 sources was equally well utilized for bone formation. Weight gains
1d tissue Mn concentration (Table 10) showed that Mn from all the com—
lunds studied was equally utilized by the pig. This study also suggests
Lat Mn requirements of young pigs for growth are not higher than 16.2
>m, based on weight gains on all the diets. The low Mn levels of
ssues from pigs on the basal diet may be a reflection of its low Mn
ntent. In this study only liver, pancreas, spleen and testes Mn
ncentrations showed a substantial response to Mn supplementation.
Hemoglobin, hematocrit, serum Mn concentration and serum alkaline
asphatase activity did not differ significantly due to source of Mn;
: serum Mn, serum alkaline phosphatase and hematocrit did differ

gnificantly (P<0.01) within the different Mn sources due to time of

 

 

 

 

 

 

55

SLE 6. ANALYSIS OF VARIANCE FOR MN SOURCE EFFECTS ON BONE AND BLOOD
PARAMETERS (SPLIT-PLOT DESIGN)

 

P—level of F—
tegory Mean Min. Max. statistic

I7

gmur, left lgg

 

 

 

 

 

Weight, g (fresh basis) 72.6 63.9 85.5 0.40
Length, cml. 9.2 8.5 9 5 0.12
External diameter,3/cm
Horizontal (B) 1.42 1.31 1.5 0.65
Vertical (D) 1.35 1.25 1.5 0.93
Internal diameter, cm
Horizontal (b) 1.02 0.81 1.31 0.89
Vertical (d) 0.98 0.80 1.20 0.79
Breaking strength, kg 165 140 187 0.67
Inertia, cm4 2 1.15 1.03 1.26 0.07
Stress at the center, kg/cm 972 856 1089 0.06
Elasticity, 1000 kg/cm2 27.6 21.6 34.2 0.33
gb, 8th left
Weight, g (fresh basis) 4.40 3.20 5.49 0.36
Ash, % of dry, fat-free rib 58.6 57.9 59.8 0.19
Specific gravity (fresh basis) 1.25 1 20 1.31 0.70
199d parameters
Serum Mn, meg/100 ml 1.65 0.91 2.71 0.28
Hemoglobin, g/100 ml 10.8 9.0 12.2 0.68
Hematocrit, % 33.4 30.2 37.7 0.70
Serum alkaline phosphatase, 7.12 5.0' 11.4 0.72

Sigma units

lMeasured from the mid—medial condyle to the fovea.

The horizontal and vertical diameters were measured at mid—
haft when the bone was positioned in such a way that the medial and
ateral condyles were facing downwards.

 

 

 

56

 

 

 

 

 

 

 

BLE 7. ANALYSIS OF VARIANCE FOR.MN SOURCE EFFECTS ON MN BALANCE,
TISSUE MN CONCENTRATIONS, ORGAN WEIGHTS AND GROWTH DATA
(SPLIT-PLOT DESIGN)

P—level of F-
ategory Mean Min. Max. statistic
alance data
Mn intake, mg/day 8.70 5.94 10.1 <0.0005
Mn excretion, mg/day

Fecal 3.82 1.77 5.63 0.001
Urinary .05 .04 .07 0.355

Mn retention, mg/day 4.84 2.52 6.15 <0.0005

Mn retention, % of intake 55.3 41.8 69.6 0.56

Mn excretion, % of intake

Fecal 44.1 29.6 57.4 0.61
Urinary .60 .39 .99 <0.0005
Qigsue, Mn, ppm (dry matter basis)
Liver 6.93 5.14 9.03 0.11
Kidney 6.60 5.55 7.87 0.12
Pancreas 4.92 3.93 6.02 0.24
Spleen 2.38 1.94 2.76 0.83
Bone 1.12 0.77 1.54 0.52

Muscle 1.84 1.50 2.22 0.22

Heart 2.51 2.33 2.77 0.80

Testes 3.88 3.22 4.39 0.82

Hair

Initial value 1.00 0.80 1.33 0.59
Final value 1.17 0.96 1.44 .22
2£gans, % of body weight

Liver 2.12 1.93 2.33 0.05

Kidney .33 .29 .37 0.35

Pancreas .14 .11 .20 0.49

Spleen .20 .14 .26 0.08

Heart .49 .44 .58 0.96

Testes .16 .10 .25 0.36

rowth data

Average daily gain, g 187 176 225 0.73

 

 

 

 

 

 

57

TABLE 8. THE EFFECT OF MN SOURCE ON THE PHYSICAL CHARACTERISTICS OF
DIFFERENT ORGANSl

 

 

 

Diet

Item Basal +MnSO4°H20 +MnCO3 +Mn0
Mn conc., ppm 16.2 26.0 24.8 27.0
No. of pigs 3 3 3 3

Abs.3 % BW'4 Abs. % BW Abs. % BW Abs. % BW SE2
Liver 324 2.10 287 1.90 311 2.209/315 2.10 .03
Kidney 51 0.34 48 0.32 47 0.33 49 0.33 .08
Pancreas 23 0.15 20 0.13 18 0.12 22 0.15 .01
Heart 80 0.53 74 0.49 70 0.48 69 0.46 .08
Spleen 32 0.21 23 0.15 30 0.21 32 0.22 .06
Testes5 34 0.22 18 0.12 20 0.13 20 0.14 .07

 

 

1Based on fresh basis.
2Statistical analyses made on percent bodyweight only.
3Absolute weight in grams.

4As percent of body weight.

5Data based on two males on diets l, 3 and 4 and 3 males on
diet 2.

6Significantly (P<0.05) greater than least value.

 

 

 

 

 

 

58

TABLE 9. THE EFFECT OF MN SOURCE ON THE PHYSICAL MEASUREMENTS OF THE
FEMURS AND RIBS, BREAKING STRENGTH AND RELATED PARAMETERS OF

 

 

 

 

 

 

THE FEMURS
Diet
Item Basal +MnSO4°H20 +MnCO3 +Mn0
Mn conc., ppm 16.2 26.0 24.8 27.0
No. of pigs 3 3 3 3
Femur, left
Weight, g (fresh basis) 73.2 77.0 68.3 71.7
Length, cmil 9.3 9.0 9.0 9.5
External diameter,Z/cm
Horizontal (B) 1.46 1.43 1.37 1.45
Vertical (D) 1.37 1.35 1.32 1.36
Internal diameter, cm
Horizontal (b) 1.05 1.03 1.03 0.96
Vertical (d) 1.05 0.94 0.94 1.00
Breaking moment, kg 171 166 166 157
Inertia, cmé 1.12 1.17 1.21 1.09
Stress at center, kg/cmz 1049 951 909 978
Elasticity, 1000 kg/cm2 27.6 24.4 30.7 27.8
Rib, 8th left
Weight, g (fresh basis) 4.9 4.6 3.8 4.5
Ash, % of dry fat-free rib 58.9 58.5 58.6 58.3
Specific gravity (fresh 1.27 1.23 1.26 1.23

basis)

 

 

 

lMeasured from.the mid-medial condyle to the fovea.

2The horizontal and vertical diameters were measured at mid-
shaft when the bone was positioned in such a way that the medial and
lateral condyles were facing downwards.

 

 

 

 

 

 

 

 

59

TABLE 10. THE EFFECT OF MN SOURCE ON THE AVERAGE DAILY GAIN (ADG) AND
TISSUE MN DISTRIBUTION

 

 

 

 

Diet
Item Basal +Mn804.H20 +MnCO3 +Mn0
Mn conc., ppm 16.2 26.0 24.8 27.0
No. of pigs 3 3 3 3
Avg. daily gain, g 200 186 180 182
Tissue Mn, ppml
Bone, 8th left rib 1.16 1.07 0.95 1.32
Liver 5.7 7.4 7.3 7.3
Kidney 6.7 6.7 6.1 6.9
Heart 2.5 2.6 2.5 2.5
Pancreas 4.5 5.1 4.8 5.3
Spleen 2.0 2.6 2.4 2.5
Testes 3.4 4.1 4.1 4.0
Muscle 1.8 2.0 1.7 1.9
Hair 1.1 1.2 1.3 1.1

 

lManganese expressed on dry matter basis except for hair and
bone. Bone Mn was expressed on dry, fat—free basis and hair on
ethanol-cleaned, air-dried basis.

 

 

2Based on 2 males on diets l, 3 and 4 and 3 males on diet 2.

 

 

 

 

 

 

 

60

sampling (Table 11). Mn retention, fecal excretion and urinary excre-
tion, all as percent of intake, showed significant (P<0.01) Mn source
effects. Time of sampling, but not Mn source, had a significant
(P<0.05) effect on urinary Mn excretion. Absolute Mn retention and
urinary Mn excretion, as percent of intake, did not differ significantly
source X time interaction. Only absolute urinary Mn excretion and
urinary Mn excretion as a percent of intake showed a significant
(P<0.05) source X time interaction.

The hematocrit value at the second sampling was significantly
(P<0.05) greater than that at the third sampling for animals receiving
supplemental MnCO3 (Table 12). Serum alkaline phosphatase activity at
the initial sampling of animals on the basal, basal supplemented with
MnSO4-H20 and basal supplemented with MnCO3 were significantly (P<0.05)
greater than at the third sampling. The hematocrit and serum alkaline
phosphatase values dropped on the second and third sampling on all
diets. Serum alkaline phosphatase levels were lowest on the basal
diet. Serum Mn increased with time of sampling on the basal diet and
basal diet supplemented with MnSOA-HZO. On diets supplemented with
MnCO3 and MnO, the levels rose on the second sampling and dropped sub—
stantially on the third.

The Mn balance data are summarized in Table 13. The low Mn intake
and excretion on the basal diet were a reflection of the lower Mn con—
tent of the basal diet as compared to the supplemented diets. The
absolute urinary excretion of Mn was the same on all diets. The abso—
lute Mn retention was significantly (P<0.01) lower on the basal diet,
essentially equal on diets supplemented with MnSO4-H20 and MnCO3, and
slightly higher on the diet supplemented with MnO. When Mn retention

was expressed as a percent of intake, the supplemented diets showed a

 

 

 

 

 

 

 

 

61

TABLE 11. THE EFFECT OF MN SOURCE AND TIME OF SAMPLING ON THE BLOOD
AND BALANCE DATAl (SPLIT-PLOT DESIGN)

 

 

 

Time of Source X time
Item Mn Source sampling interaction
Serum alkaline phosphatase 0.72 <0.0005 0.41
Serum Mn 0.48 0.007 0.40
Hemoglobin 0.68 0.16 0.09
Hematocrit 0.70 0.001 0.59
Mn intake <0.0005 0.009 0.28
Mn excretion, absolute
Fecal 0.001 0.43 0.82
Urinary 0.36 0.028 0.03
Mn retention, absolute <0.0005 0.38 0.87
Mn retention, % of intake 0.56 0.23 0.59
Mn excretion, % of intake
Fecal 0.61 0.23 0.59
Urinary <0.0005 0.13 0.03

 

 

1P—level of F-statistic.

 

 

 

 

62

TABLE 12. EFFECT OF MN SOURCE AND TIME OF SAMPLING ON SOME BLOOD

 

 

 

PARAMETERS
Diet
Item Basal +MnS04°H20 +MnCO3 +Mn0
Mn conc., ppm 16.2 26.0 24.8 27.0
NO. of pigs 3 3 3 3
Samplingel/ 1 2 3 1 2 3 1 2 3 1 2 3

 

Hemoglobin, ' 10.9 10.4 10.2 11.1 10.1 11.2 11.0 10.7 10.6 10.9 11.3 10.9
g/100 ml '

I
i
L
l

Hematocrit, 33.7 33.4 31.8%34.9 33.7 32.8f33.3 34.1 31.4§34.1 33.8 32.9
2 : i .g/ ‘

Serum alkaline i -

phosphatase, 7.9 6.0 5.3. 9.3 6.4 6.3: 9.0 6.8 6.3 8.1 7.3 7.2

Sigma units _2_/ ; E/ ;_ _2_/

Serum Mn, mcg/ 1.1 1.2 1.3. 1.5 1.6 1.7? 2.0 2.1 1.5' 2.0 2.9 1.9
100 ml 5 _g/

1Blood sampling weekly.

 

2Values under the same dietary source significantly (P<0.05) dif-
ferent than the least value.

 

 

 

 

 

 

 

 

 

 

 

63

TABLE 13. RETENTION AND ROUTES OF EXCRETION 0F MN FROM BASAL DIET AND
BASAL DIET SUPPLEMENTED WITH MANGANOUS SULFATE (MnSO4-H20),
MANGANOUS CARBONATE (MnCO3) OR MANGANOUS OXIDE (MnO)

 

 

 

 

Diet
Item Basal +MnSO4'H20 +M'nCO3 +Mn0
Mn conc., ppm 16.2 26.0 24.8 27.0
No. of pigs 3 3 3 3
Mn intake, mg/day 6.0 9.62/ 9 22/ 10.0fl/
Mn excretion, mg/day l/ 1/
Fecal 2.7 4.2- 3.8 4.2—
Urinary 0.1 0.1 0.1 0.1
Mn retention, mg/day 3.2 5.32! 5.32] 5.7g/
Mn retention, % of intake 53.4 55.1 57.4 57.0
Mn excretion, % of intake
Fecal 44.94/ 43.8 41.3 41.9
Urinary 1.7- 1.0 1.1 1.0

 

1Significantly (P<0.05) greater than least value.
2Significantly (P<0.01) greater than least value.
3Significantly (P<0.01) greater than least two values.

4Significantly (P<0.01) greater than least three values.

 

 

 

 

 

 

 

 

 

 

 

64

slight advantage over the basal diet. Mn from MnSOA-HZO was retained
to a lesser extent than that from the other two compounds. Fecal Mn
excretion as a percent of intake was slightly higher on diets supple—
mented with MnSO4°H20 than those supplemented with MnCO3 and MnO.
Urinary Mn excretion as a percent of intake was significantly (P<0.01)
different between the basal and the supplemented diets, but not within
the supplemented diets. Fecal and urinary Mn excretion, both as a
percent of intake, on the basal diet were higher than that on the
supplemented diets. Regardless of dietary Mn source, over 90% of the

excreted Mn was found in the feces.

3. Mn Availability Studies Using a Replicated Latin Square Desigp
(where all animals were subjected to all experimental diets in four
collection periods)

The Mn source had no significant effect on the blood parameters
(Table 14). There was a slightly lower serum Mn on the basal diet,
presumably as a reflection of the lower Mn content of the diet. All
sources were equally effective in maintaining the levels of hemoglobin,
hematocrit and serum alkaline phOSphatase (Table 15). Mn retention,
fecal and urinary Mn excretion, all as percent of intake, were signifi-
cantly (P<0.01) different between treatments (Table 16). Mn intake and
absolute fecal Mn excretion and retention were significantly (P<0.01)
different, but not absolute urinary Mn excretion.

The absolute fecal Mn excretion on the basal diet was significantly
(P<0.01) lower than on the supplemented diets. Absolute fecal Mn excre-
tion was slightly higher on diets supplemented with MnSO4'H20 and MnO
than those supplemented with MnC03. The absolute urinary Mn excretion

was equal on all diets but, when expressed as a percent of intake, the

 

 

 

 

 

 

 

 

 

 

 

 

65

EFFECTS OF MN SOURCE 0N BLOOD AND MN BALANCE MEASURES

 

 

 

 

TABLE 14.
(REPLICATED LATIN SQUARE DESIGN)
P—level of F—
Item Mean Min. Max. statistic
Bloodgparameters
Hemoglobin, g/100 ml 10.74 8.17 14.92 0.181
Hematocrit, % 33.95 31.00 44.80 0.528
Serum alkaline phosphatase, 7.42 4.70 11.40 0.559
Sigma units '
Serum Mn, meg/100 ml 1.94 1.26 3.34 0.160
Balance data
Mn intake, mg/day 7.08 4.65 9.41 <0.0005
Mn excretion, mg/day
Fecal 3.63 2.54 4.98 <0.0005
Urinary .030 .008 .067 0.264
Mn retention, mg/day 3.39 1.49 5.72 <0.0005
Mn retention, % of intake 47.37 30.00 67.02 <0.0005
Mn excretion, % of intake
Fecal 52.14 32.82 69.11 <0.0005
.14 .39 .88 <0.0005

Urinary

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 15. EFFECT OF MN FROM DIFFERENT SOURCES ON SOME BLOOD MEASUREMENTS

 

 

 

Diet
Item Basal +MnSO4°H20 +MnC03 +Mn0
Mn conc., ppm 16.2 26.0 24.8 27.0
NO. of pigs 12 12 12 12
Hemoglobin, g/100 ml 11.1 10.4 10.9 10.6
Hematocrit, % 34.5 34.3 33.7 33.4
Serum alkaline phosphatase, 7.56 7.39 7.67 7.06
Sigma units
Serum Mn, mcg/100 ml 1.70 2.15 1.76 2.25

 

 

 

 

 

 

 

 

 

 

67

 

 

 

 

TABLE 16. RETENTION AND ROUTES OF EXCRETION OF MN FROM BASAL DIET AND
‘ BASAL DIET SUPPLEMENTED WITH MANGANOUS SULFATE (MnSO4°H20),
MANGANOUS CARBONAIE (MnCOB) AND MANGANOUS OXIDE (MnO)
Diet
Item Basal +Mn804°H20 +MnCO3 +Mn0
Mn conc., ppm 16.2 26.0 24.8 27.0
No. of pigs 12 12 12 12
Mn intake, mg/day 5.17 8.315! 7.933/ 8.639]
MnF:::Ietion, mg/day 3.10 4.26g/ 3.912/ 4.242!
Urinary 0.030 0.034 0.030 0.028
Mn retention, mg/day 2.04 4.02%] 3.99g/ 4.36;]
Mn retention, % of intake 38.8 48.0i/ 50.1g/ 50.3g/
Mn excretion, Z of intake 4/5/
Fecal 60.6—-— 51.6 49.5 49.4
Urinary 0.56é/ 0.39 0.36 0.31
lSignificantly (P<0.05) greater than least value.
2Significantly (P<0.01) greater than least value.
3Significantly (P<0.05) greater than least two values.
4Significantly (P<0.01) greater than least two values.
5Significantly (P<0.05) greater than least three values.
6Significantly (P<0.01) greater than least three values.

 

 

 

 

 

 

68
pigs on the basal diet excreted significantly (P<0.01) more urinary Mn
than the pigs on supplemented diets. Within the supplemented diets,
urinary Mn excretion was not significantly different. Absolute Mn
retention was significantly (P<0.01) higher on the supplemented diets
than on the basal diet. Mn from the diet supplemented with MnO was
retained in somewhat greater amounts than Mn from those diets supple-
mented with M'nCO3 and MnS04~H20, but these differences were not statis—
.tically significant. When expressed as a percent of intake, Mn retention
on the basal diet was significantly (P<0.05 or P<0.0l) lower than that
on supplemented diets. Fecal Mn excretion, as a percent of intake, on
the basal diet was significantly (P<0.05 or P<0.01) higher than that
on the supplemented diets, but the differences between the supplemented
diets were not significant. Irrespective of Mn source, the main route

of Mn excretion was fecal, and urinary Mn excretion was very small and

constant on all diets.

4. Discussion of the Results of Experiment 1

The low Mn retention on the basal diet and the high Mn excretion
on the same diet agrees with the findings of Mbrimoto et a2. (1959),
who showed poor Mn availability to chicks when soy protein was used in
the diet, and of Davis at al. (1962), who reported that soy protein
contained a component which combined with Mn, making it unavailable.
In this particular study, most of the Mn in the basal diet was supplied
by soy protein. The dominance of the fecal route as a means of Mn excre-
tion and the small but constant urinary Mn excretion found in this study
have been reported by others in other species (Underwood, 1971; Thomas,
1970; Miller, 1973). The 40 to 50% Mn retention by the pig shown in

this study parallels Mn retention in the human reported by North at al.

 

 

 

 

 

 

69
(1960). Brown and MtCracken (1965) found that chickens retained 32%
of the absorbed Mn.

The conclusion that Mn in MnSO4-H20, MnCO3, and MnO is equally
available to a pig for growth agrees with the findings in other species.
Schaible et a1. (1938) and Gallup and Norris (1939) found that the
sulfate, carbonate and oxide were equally effective in preventing
perosis in chickens. Cotzias and Greenough (1958) and Zajvec (1959)
have reported that the absorption of Mn was not affected by its valency
state in the compounds used. But more recently others have reported
that Mn oxides are not as available as the sulfate, carbonate and
chloride (Anke at al.. 1967; watson at al.. 1970, 1971).

Serum alkaline phosphatase and serum Mn levels were equal on all
diets, which probably indicates that Mn from all sources was equally
available to the growing pig or that the levels of Mn provided in all
diets were high enough to sustain normal levels of serum alkaline phos-
phatase and serum Mn. Mn deficiency has been shown to reduce serum
alkaline phosphatase and serum Mn levels in swine as well as other
species (Plumlee at aZ., 1956; Rojas at al., 1965; Swaney and Kehar,
1958; Hawkins et aZ., 1955; Ugnenko, 1972). The effect of time of
sampling (age of the pig) on hematocrit and serum alkaline phosphatase
values found in this study is supported by the findings of Miller at
al. (1961), who showed a similar drop at a similar age of pigs. Long
at al. (1965) also reported high serum alkaline phosphatase values in
young pigs which gradually dropped with age.

In this study, liver, testes, and spleen Mn concentration increased
with dietary Mn levels, which is in agreement with the reports of
Leibholz at al. (1962), Johnson (1943, 1944), Grummer et al. (1950),

and Underwood (1971). The response of spleen to Mn supplementation has

 

 

 

 

 

 

 

70

been shown in rats by Ugnenko (1972). Hair and bone, which were
reported to respond to Mn supplementation by Leibholz at al. (1962),
did not do so in this study. This may be due to the fact that Leibholz
at al. (1962) used a 100-fold margin between the basal diet and the
supplemented diet as compared to a twofold margin used in this study.
B. Experiment 2: Study of the gastrointestinal flux pattern of Mn

from.different Mn sources using chromic oxide (Cr203) as an

indicator

1 The results of this study are summarized in Tables 17, 18 and 19
and Figures 2.1 through 2.4. The cranial small intestine was a major
route of absorption for Mn from the supplemented diets but not for Mn
from the basal diet. There was no net Mn absorption by the caudal small
intestine. Hendricks (1967) showed that pigs fed a 16% soy protein diet
supplemented wtih Mn from Mn804°H20 displayed net absorption in the
stomach and cecum only. Since there is a large amount of Mn in the
bile (Kent and McCance, 1941; Mahoney and Small, 1968; Starodubova,
1968), and bile is screted into the cranial small intestine, it is
apparent that in the case of Mn from the supplemented diets, the cranial
small intestine must be absorbing Mn faster than it is being secreted
into this section of the gut. This finding makes the cranial small
intestine a very important and efficient homeostatic mechanism for
regulating Mn levels in the body. Britton and Cotzias (1966) concluded
that it is the variable excretion rates rather than regulated absorp—
tion that seem to maintain constant tissue Mn levels. Howes and Dyer
(1971) and Miller (1973) reported that absorption differences are the
main homeostatic control mechanisms for body Mn levels.

Although digesta pH did not differ significantly between treatments

(Table 18), there was a substantial treatment pH difference in the

 

 

 

 

 

71

TABLE 17. NET ABSORPTION AND SECRETION OF MN IN THE DIFFERENT SECTIONS
OF THE GUT BY THE GROWING PIG FED DIFFERENT MN SOURCESl

 

 

 

Diet
Basal +MnSO4°H20 +MnCO3 +Mn0

Section of gut Abs.2 Sec.3 Abs. Sec. Abs. Sec. Abs. Sec.
Stomach 6O 61 49 52
Small intestine4

Cranial 129 22 62 60

Caudal 185 560-51 8799/ 9679/
Cecum 52 41 19 34
Colon 39 33 24 12
Rectum 64 138 13 119

 

lNet flux as percent of Mn in feed or digesta in previous gut
section.

 

2Net absorption.

3Net secretion.

4Divided roughly in two halves.

5Significantly (P<0.01) greater than least value.

6Significantly (P<0.01) greater than least two values.

 

 

 

L.

 

 

TABLE 18. GUT CONTENT WEIGHTS AND le

 

 

 

 

Diet
Section of gut Basal +MnSO4°H20 +MnCO3 +Mn0
pH values
Stomach 2.75 3.36 3.19 3.07
Small intestine
~Cranial 6.52 6.25 5.59 6.40
Caudal 6.61 6.13 6.77 6.70
Cecum 5 97 '6 38 5.80 6 l4
Colon 6.09 6 19 6.08 6 10
Rectum 6.45 6.42 6.39 6.39
Gut contents, g (wet basis) 5/
Stomach 459— 348 351 331
Small intestine
Cranial 314 347 323 292
Caudal 361 3234/ 330 3583/
Cecum 442 498- 372 458'-~
Colon 74 91 163 125
Rectum 72 81 62 61

 

 

1Based on three animals per treatment.

2Divided roughly into two halves.

3Significantly (P<0.05) greater than least value.
4Significantly (P<0.001) greater than least value.

5Significantly (P<0.05) greater than least three values.

 

 

 

 

73

TABLE 19. MEAN, MINIMUM AND MAXIMUM VALUES OF pH, WET GUT CONTENTS AND
NET ABSORPTION AND SECRETION IN DIFFERENT SECTIONS OF THE GUT

 

 

 

 

 

 

P-level of F-
Item Mean Min. Max. statistic
BE
Stomach 3.09 1.88 3.88 0.82
Small intestine
Cranial 6.21 5.02 7.20 0.29
Caudal 6.55 5.36 7.34 0.39
Cecum 6.07 5.38 6.83 0.50
Colon 6.12 5.72 6.38 0.92
Rectum 6.41 6.05 6.90 0.99
Gut contents, g
Stomach 372 286 496 0.01
Small intestine
Cranial 319 265 386 0.25
Caudal 343 278 405 0.75
Cecum 113 42 182 0.02
Colon 443 285 553 0.13
Rectum 69 50 93 0.21
Net absorption (+) or secretion (-)1
Stomach 56 41 78 0.63
Small intestine
Cranial 6 -224 78 0.01
Caudal —648 -1176 2 0.02
Cecum 36 5 73 0.06
Colon —21 -85 22 0.21
Rectum ~1002 ~264 —l 0.24

 

lNet flux as percent of Mn in feed or digesta in previous
gut section.

2Divided roughly into two halves.

 

 

 

 

 

 

 

74

Figure 2.1. Summary of net Mn flux from sections of the gastro-
intestinal tract of the growing pig fed the basal diet.

 

 

75

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76

 

Figure 2.2. Summary of net Mn flux from sections of the gastro- ‘
intestinal tract of the growing pig fed the basal diet supplemented i
with MnSO4 ' H20 . 1|

 

 

77

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78

 

Figure 2.3. Summary of net Mn flux from sections of the gastro-
intestinal tract of the growing pig fed the basal diet supplemented
with MnCO3.

 

 

 

 

 

79

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80

 

 

Figure 2.4. Summary of net Mn flux from sections of the gastro-
intestinal tract of the growing pig fed the basal diet supplemented
with MnO.

 

 

 

 

 

 

 

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82
cranial small intestine, being more acid in the case of pigs fed the
supplemented diets than those fed the basal diet (6.08 vs 6.52). This
might account for the differences in absorption rates. It could be
postulated that the Mn absorption mechanisms might be more efficient at
a lower pH in the cranial small intestine than at a higher pH. Man—

ganese from the basal diet is presumably held in some form which makes

 

it less available for absorption in this region of the gut. Davis et
a2. (1962) reported a factor in soybean which tends to tie up Mn and
make it unavailable. Most of the Mn in the basal diet was supplied by
soy protein.

In Experiment 1 it was shown that when soybean meal and corn were
the sole sources of Mn, there was a relatively high fecal Mn excretion

and a low Mn retention as a percent of intake, plus low tissue Mn con—

 

centrations. This indicated a lower net absorption of Mn from the
basal diet, perhaps due in part to the fact that net absorption of Mn
from the basal diet occurred only in the stomach and cecum.

The net absorption of Mn from the basal diet (feed sources) in
the stomach was slightly higher than that of Mn from the supplemented
diets, but in the cecum the net absorption of Mn from the basal diet
was much higher than that of Mn from the supplemented diets. The gut
fill in the stomach and cecum was significantly (P<0.05) different
between treatments. The stomach contents of the animals on the basal
diet were significantly (P<0.05) heavier than those of the animals on
other diets. The wet weight of the cecal contents of animals on the

diets supplemented with MnSO O and MnO were significantly (P<0.05 or

4'H2
P<0.01) heavier than those of animals on the basal diet (Table 18).
Within the supplemented diets, the net absorption of Mn from the diet

Supplemented with MnSO4~H20 was highest and that from the diets

 

 

 

 

83
supplemented with MnCO3 lowest in both the stomach and cecum. In the
cranial small intestine, the net absorption of Mn from the diets sup-

plemented with MnCO was highest while that from the diets supplemented

3
with MnSO4'H20 was lowest. The net absorption of Mn from diets sup—
plemented with MnO was intermediate in the stomach, cranial small
intestine and cecum.

The net secretion of Mn was significantly (P<0.01) higher on the
supplemented diets when compared to the basal diet in the caudal small
intestine, slightly higher in the rectum but slightly lower in the
colon. Within the supplemented diets, the net Mn secretion was sig—
nificantly (P<0.01) higher on diets supplemented with MnO and MnCO3
than on diets supplemented with MnSO4°H20 in the caudal small intestine
(Table 17). The net Mn secretion on diets supplemented with MnO or
MnCO3 was not significantly different. Net Mn secretion in the rectum
was much greater on diets supplemented with mnsoé-Hzo and MnO than on
the diets supplemented with MnCO3. In the colon, the net Mn secretion
was highest on diets supplemented with MnSO4°H20 and lowest on diets
supplemented with MnO. Hendricks (1967) reported net Mn secretion into
the cranial small intestine, caudal small intestine, and colon when
pigs were fed a 16% soy protein diet supplemented with Mn from
MnSO4'H20. He showed a high net Mn secretion in the colon, a low net
Mn secretion in the cranial and caudal small intestine, and an absence
of Mn secretion in the rectum.

C. Experiment 3: The effect of high level Ca and P supplementation
and an inverse Ca-P ratio on Mn utilization by the growing pig

 

The effects of different Ca to P ratios, Ca and P levels and Mn
supplementation on physical and chemical composition of pig tissues are
Summarized in Tables 20, 21, and 22. A 2 to 1 ratio of Ca to P sig-

nificantly (P<0.05) depressed rib Mn content and slightly increased

 

 

 

 

 

 

 

84
heart, pancreas and serum Mn as compared to a l to 2 ratio. The
increased levels of Ca and P supplementation significantly (P<0.01)
increased rib and pancreas Mn concentration but also significantly
(P<0.01) depressed Mn concentration of the metacarpal bone. The high
levels of Ca and P slightly increased heart Mn and significantly
(P<0.05) increased serum Mn concentration. This finding is at variance
with those of Hawkins at al. (1955), who reported a suppression of
serum Mn with high Ca and P intakes in cattle. Lassiter at al. (1970)
reported that rats given a 0.9 percent P in the diet caused signifi-

54Mn than did 0.4 percent P.

cantly higher retention of orally administered
Mn supplementation increased heart Mn significantly (P<0.01) and
slightly increased rib, pancreas and serum Mn, kidney Ca and serum inor-
ganic P. High Mn levels in the diets significantly (P<0.05) depressed
rib Ca and Mg and slightly depressed metacarpal Mn, P and Mg, serum Ca,
and rib and kidney P. The slight increase of Mn in serum following
increased Mn intakes is in harmony with other observations in swine
(Plumlee at al., 1956), and with some reports in cattle (Rojas et al..
1965; Hawkins et al., 1955), in rats (Ugnenko, 1972), and in poultry
(Bolton, 1955). Other reports, however, are in contrast to these find—
ings (Krieg, 1966; Bentley and Phillips, 1951a). In poultry, Nielsen
and Madsen (1942) observed no appreciable difference in blood Ca concen—
tration due to dietary Mn levels, and reported that acid soluble P of
the blood and inorganic P of plasma did not differ significantly due to
Mn supplementation.
Although Mn has been implicated in bone formation (Underwood, 1971),
the results of this study showed no significant effect of Mn supplemen-

tation on rib and metacarpal physical measurements, breaking strength

and related parameters (Tables 20 and 21). Dietary Mn levels did not

 

 

 

 

 

 

 

85

TABLE 20. EFFECT OF THE CA-P RATIO, CA AND P LEVELS AND MN SUPPLEMENTA—
TION ON PHYSICAL AND CHEMICAL PARAMETERS OF THE FIRST, LEFT

 

 

 

 

 

RIB
Ca-P ratio Ca—P level Suppl. Mn, ppm
Item 0.5 2.0 1X 2X 0 40 SE
Physical measurements 2/ 1/
Weight, g (fresh 12.5—- 10.9 9.4— 14.0 11.4 12.0 0.36
basis)
External diameter 1.71 1.66 1.60;! 1.77 1.70 1.67 0.04
(B) . cm
Internal diameter .581] .51 .57 .52 .56 .53 0.02
(d) . cm
Specific gravity 1.22 1.21 1.15;! 1.27 1.22 1.21 0.01
(fresh basis)
Inertia, cm4 .24 .22 .18}! .28 .24 .21 0.02

1/

Breaking moment, kg 26.8 28.8 15.1- 40.5 28.4 27.2 1.04

Chemical measurements

Ash content, Z 56.9 52.9“ 50.5 59.3 55.5 54.3 0.42
Calcium, Z 23.0l/ 21.7 20.51! 24.2 22.6l/ 22.0 0.18
Phosphorus, Z 10.9l/ 9.9 9.51! 11.2 10.5 10.3 0.09
Magnesium, Z .36}! .31 .30 .36 .35}! .32 0.01
Manganese, ppm 1.07;! .99 .951! 1.11 1.02 1.04 0.03

 

1Numbers on the same line under the same subheading, i.e., Ca-P
ratio, Ca-P level, or Mn supplementation level, are significantly
(P<0.05) different.

2P<o.01.

3Expressed on dry, fat-free bone.

 

 

 

TMME 2‘

[tan

Physical
Weight

bas:

Exteri
(B) .

Inter!

(d):

Specii
(fr¢

Inert:
Break:
9131222;
Ash c<
Calcit
Phospl
Hague
Manga

\‘

ratio,
(P<0.05

 

 

 

85

 

 

 

 

 

TABLE 20. EFFECT OF THE CA—P RATIO, CA AND P LEVELS AND MN SUPPLEMENTA-
TION ON PHYSICAL AND CHEMICAL PARAMETERS OF THE FIRST, LEFT
RIB
Ca—P ratio Ca—P level Suppl. Mn, ppm
Item 0.5 2.0 1X 2X 0 40 SE
Physical measurements 2/ 1/
weight, g (fresh 12.5- 10.9 9.4— 14.0 11.4 12.0 0.36
basis)
External diameter 1.71 1.66 1.60;] 1.77 1.70 1.67 0.04
(B) . cm
Internal diameter .58}! .51 .57 .52 .56 .53 0.02
(d) . cm
Specific gravity 1.22 1.21 1.15;! 1.27 1.22 1.21 0.01
(fresh basis)
Inertia, em4 .24 .22 .181! .28 .24 .21 0.02
1/

Breaking moment, kg 26.8 28.8 15.}—

40.5 28.4 27.2 1.04

Chemical measurements

 

Ash content, Z 56.9 52.9 50.5 59.3 55.5 54.3 0.42
Calcium, Z 23.0£/ 21.7 20.51] 24.2 22.6l/ 22.0 0.18
Phosphorus, Z 10.9l/ 9.9 9.5;, 11.2 10.5 10.3 0.09
Magnesium, z .36l/ .31 .30 .36 .351/ .32 0.01
Manganese, ppm 1.07;! .99 .951! 1.11 1.02 1.04 0.03

 

1Numbers on the same line under the same subheading, i.e., Ca-P
ratio, Ca—P level, or Mn supplementation level, are Significantly

(P<0.05) different.

2

P<0.01.

3Expressed on dry, fat-free bone.

 

 

In

rat
diI

era

 

 

 

86

TABLE 21. EFFECT OF THE CA-P RATIO, CA AND P LEVELS AND MN SUPPLEMENTA~
TION ON PHYSICAL, CHEMICAL AND HISTOPATHOLOGICAL PARAMETERS

OF THE 5TH METACARPAL

 

Ca—P ratio Ca—P level Suppl. Mn, ppm
Item 0.5 2.0 1X 2X 0 40 SE

 

Physical measurements 2/
Weight, g (fresh 14.6 13.9 12.8- 15.8 14.4 14.1 0.39
basis)
External diameter 1.38 1.38 1.332] 1.43 1.39 1.37 0.02
(D). cm
External diameter 1.36 1.32 1.28%] 1.40 1.35 1.34 0.02
(B). cm
Internal diameter .97 .96 .94 .99 .991] .94 0.02
(b), cm
Specific gravity 1.23 1.21 1.18;! 1.25 1.22 1.21 0.01

(fresh basis)

 

 

 

Inertia, an4 1.04 1.12 .873! 1.30 1.08 1.09 0.09
Breaking moment, kg 42.6 43.0 29.22! 56.3 44.3 41.3 1.80
Stress, kg/cm2 299 263 2292/ 333 292 270 20.01
Elasticity, 2 13.72! 7.3 10.4 10.7 10.6 10.5 0.57
1000 kg/cm
Chemical measurementsg/ 2/ 2/
Ash content, Z 58.157 54.5 52.737 59.9 56.8 55.8 0.57
Phosphorus, Z 10.2- 9.4 9.1-2/10.5 9.9 1/ 9.8 0.12
Manganese, ppm .992/ 1.04 1.095] .94 .96— 1.07 0.03
Magnesium, Z .34— .29 .29- .34 .32 .30 0.01
Bone histology 2/ 1/
Epiphysis 4] 1.50 1.63 1.31- 1.81 1.75- 1.38 0.12
Thickness of compact 2/ 2/ 1/
bone of the 96.2— 51.8 45.8— 102.3 91.6- 56.4 10.59
diaphysis 2!
Thickness of epiphyseal 2/ _
cartila enous 65.6 64.1 70.3—- 59.4 62.5 67.2 2.33
plate 2

 

1Numbers on the same line under the same subheading, i.e., Ca-P
ratio, Ca-P level, or Mn supplementation level, are significantly (P<0.05)

different.

21><o.01.

3Expressed on dry, fat-free bone.

41 = normal; 2 = very slight change; 3 = slight change; 4 = mod-
erate change; 5 = severe change.

5Percent of a 20X objective by 10X ocular field.

 

 

 

 

 

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88

affect Ca and P metabolism in the bones, which is in accord with the
findings of Parker at al. (1955) that Mn intake did not affect the
quantities of radioactive Ca and P deposited in the bones of chicks.
Ellis at al. (1947) reported no significant difference in breaking
strength or Ca content of the humerus of rabbits due to dietary Mn
levels, but this report is contrary to the reports of Smith at al.
(1944), who showed a significant decrease in weight, density, length,
breaking strength and ash content of the ulna and humerus of rabbits
due to dietary Mn levels. Hemoglobin and hematocrit values were not
affected by Mn supplementation. Sawney and Kehar (1958) reported
similar findings in cattle. Similarly Mn supplementation did not sig—
nificantly affect serum Ca, inorganic P, Mg, Mn and alkaline phosphatase.
This work is in accord with the findings of Hawkins at al. (1955),
Fain at al. (1952) and Sawney and Kehar (1958) in cattle, but Hawkins
at al. (1955) and Fain et a1. (1952) showed a depression of serum Mg
due to Mn supplementation.

The slight increase in serum alkaline phosphatase activity due to
Mn supplementation has also been reported by some (Lassiter at aZ.,
1970) but not others (Leibholz et al., 1962). There was a time of
sampling effect on serum Ca, inorganic P, Mn, Mg and alkaline phospha—
tase. In general, their levels increased within the first month except
in the case of P, which declined drastically throughout the experimental
period on diets 5 and 6 with low P content (0.35Z) derived exclusively
from corn and soybean meal. The serum Mg values tended to be higher
on these same diets than on other diets.

A two—way interaction between ratio and level of Ca and P on Mn
metabolism is shown in Tables 23 and 24. This interaction was signifi—

cant (P<0.01 or P<0.05) relative to levels of serum, liver and pancreas

 

 

Chang

 

 

89

EFFECT OF THE INTERACTION BETWEEN CA~P RATIO AND LEVEL OF CA

 

 

 

 

TABLE 23.
AND P ON MN METABOLISM AND SOME OTHER BONE PARAMETER85
Ca-P Ratio
0.5
Ca—P level Ca/P level
Item 1X 2X 1X 2X SE
Ribz lst left 2/
Weight, g (fresh basis)—- 11.6 13.4 7.2 14.7 0.51
External diameter (D), cur- .75 .72 .65 .76 0.03
Specific gravityE/ (fresh 1.21 1.24 1.10 1.31 0.01
basis) 4 2/
Inertia, cm —- 2/ .22 .26 .13 .31 0.02
Breaking moment, kr- 23.0 30.7 7.3 50.2 1.60
Ash content 7.2/ 55.1 58.6 45.9 59.9 0.59
Calcium, Z£7 / 22.1 23.8 18.9 24.5 0.26
Phosphorus, Z— 10.6 11.1 8.4 11.3 0.13
Magnesium, 22 .39 .34 .22 .39 0.01
Metacarpalz 5th
Weight, g. (fresh basis)2/ 14.8 14.4 10.7 17.2 0.56
External diameter (D), cm? 1.39 1.36 1.27 1.49 0.03
External diameter (B), cnr- 1.36 1.37 1.20 1.44 0.03
Specific gravity_2_ (fresh 1.22 1.23 1.14 1.28 0.01
basis) /
Inertia, cm - 2/ 1.16 .93 .58 1.67 0.13
Breaking moment kg— 41.7 43.4 16.7 69.3 2.55
Ash content Z.) 57.1 59.1 48.2 60.7 0.81
Calcium, 7.27 2/ 21.0 20.8 18.2 21.2 0.71
Phosphorus, Z— 10.1 10.4 8.1 10.7 0.16
Magnesium, Z1. .36 .31 .22 36 0.01
Manganese, ppnr- 2 2/ 1.00 .98 1.18 .91 0.04
Elasticity, 1000 kg/cm —- 15.33 12.12 5.44 9.17 0.81
Bone histology
Diaphysis 3/1/ 1.38 1.25 1.13 2.13 0.24
Epiphysis 3/2/ 1.63 1.00 1.38 2.25 0.17
“ 65.63 75.00 65.63 64.06 3.29

Thickness of epiphyseal
cartilagenous plate 4/2/

1

2P<0.01.

31=norma1; 2=very slight change; 3=slight change; 4=moderate

change; 5=severe change.

4Percent of a 20K objective by 10X ocular field.

Significant (P<0.05).

5Chemical constituents expressed on dry, fatefree bone.

 

I): l 53.

m

 

9O

EFFECT OF THE INTERACTION BETWEEN CA-P RATIO AND LEVEL OF CA

 

 

 

TABLE 24.
AND P ON ORGAN AND SERUM MN METABOLISM, AND SOME OTHER
MEASURES
Ca—P Ratio
0.5 " 2.0
Ca—P level Ca/P level
Item 1X 2X 1X 2X SE
Organ chemical composition
(dry matter basis)
Liver Mn, ppm. 13.76 13.42 12.93 15.50 0.58
Kidney P, zl/ / 1.39 1.31 1.37 1.43 0.03
Pancreas Mn, ppur- 5.41 6.66 5.99 6.23 0.23
Serum parameters
Serum alkaline phosphatase
(Sigma units) /
lst bleeding- 5 88 5.00 4.45 6.20 0.34
2nd bleedingf 5 08 6.83 7.98 6.15 0.39
3rd bleeding— / 4.75 5.25 7.40 5.08 0.38
Serum Mn, meg/100 m1—
3rd bleeding 2.29 2.89 3.21 2.47 0.10
Serum inorganic P mg/100 ml
lst bleeding_] 9.7 10.1 13.5 10.7 0.01
3rd bieedingi/ 10.4 9.0 6.4 8.0 0.04

 

 

 

lSignificant (P<0.05).

2P<0.01.

 

int

con
.i
b
s

 

 

91
Mn, and metacarpal Mn levels. High levels of Ca and P in a 1 to 2
ratio depressed pancreas Mn content appreciably and slightly depressed
liver and metacarpal Mn content, but increased serum Mn. High levels
of Ca and P supplementation in a 2 to 1 ratio increased liver and
pancreas Mn but depressed the serum and metacarpal Mn concentration.
Hawkins et al. (1955) reported that high intakes of Ca and P tended
to suppress blood Mn.

The interaction between the ratio of Ca to P and Mn level on Mn
and Mg metabolism is shown in Table 25. Feeding Mn along with Ca and
P in a l to 2 ratio slightly depressed liver Mn but increased it when
Ca and P were given in a 2 to 1 ratio. Although Mn supplementation
alone did not significantly affect metacarpal Mg, feeding Ca and P in
a l to 2 ratio did increase metacarpal Mg significantly (P<0.05). The
interaction between Ca to P ratio and diet Mn level upon serum Mn
concentration was not significant, contrary to Hawkins' et al. (1955)
findings in cattle.

The interaction between diet Ca and P levels and Mh 18V81S on some
bone and serum parameters is shown in Table 26. This interaction was
significant (P<0.05) relative to rib ash content, Ca and P, metacarpal
Mn levels and serum inorganic P. With low Ca and P levels, Mn supple-
mentation increased metacarpal Mn and serum inorganic P bUt depressed
rib ash, Ca and P content. Mn supplementation, coUPled With high Ca and
P levels, did not affect metacarpal Mn, slightly increased rib aSh’ Ca

' t
and P, but depressed serum inorganic P. The interaction between dle

1
Ca and P levels and Mn levels was not Significant relative to Mg leve S

' th
in the tissues. The significant effect of Mn supplementation on e

levels was
metacarpal internal vertical diameter, rib Mg and heart Mn

h diets.
SUppressed when high Ca and P levels were also given in t e

 

92

TABLE 25. EFFECT OF THE INTERACTION BETWEEN CA—P RATIO AND MN LEVELS
0N MN AND MG METABOLISM

 

 

 

 

 

Ca-P ratio
0.5 2.0
Mn-level Mn-level

Item 0 40 0 40 SE
Metacarpalz 5th left

Mg, ‘4 y .36 .31 .29 .29 0.01
.Liver / 13.8 13.4 12.9 15.5 0.58

Mn. ppm-
Bone histology

Epiphysis 2/1/ 1.88 1.63 1.13 1.63 0.17

 

lSignificant (P<0.05).

21 = normal; 2 = very slight change; 3 = slight change; 4 =
moderate change; 5 = severe change.

 

 

18“

 

93

TABLE 26. EFFECT OF THE INTERACTION BETWEEN CA-
ON SOME PHYSICAL PARAMETERS

P LEVELS AND MN LEVELS

 

 

 

 

 

 

Ca—P level

 

 

 

 

 

 

W
Mn—level Mn-level
Item 0 40 T70“ SE
Rib, lst left /
Ash content, %— 51.81 49.13 59.14 59.42 0.59
Calcium, 7.1] / 21.08 19.86 24.17 24.18 0.26
Phosphorus, Z—- 9.77 9.31 11.15 11.22 0.13
Metacarpal, 5th left
Manganese, ppm 1] .98 1.19 .94 .94 0.04
Serum inorga ic P,
mg/100 ml;
3rd bleeding 7.48 9.25 9.00 7.98 0.60
Bone histology
Thickness of epiphyseal
cartilagenous plateE/I/ 71.88 53.13 68.75 65.63 3.29

 

lSignificant (P<0.05).

2Percent of a 20X objective by 10X ocular field.

sea.‘
i
We

diet.

emu

reBard.

 

 

 

94

Table 27 summarizes a three-way interaction between level of Ca
and P, ratios of Ca to P and Mn levels on some measured parameters.
The interaction was significant (P<0.05) with respect to rib and serum
Mn content, pancreas dry matter, and metacarpal Mg content and elasti—
city. With low Ca and P levels in a l to 2 ratio, Mn supplementation
increased serum and metacarpal Mn values, slightly increased pancreas
dry matter and did not change metacarpal Mn and elasticity. High Ca
and P levels in a 1 to 2 ratio with Mn supplementation depressed rib
Mn, metacarpal Mg, pancreas dry matter and serum Mn, but increased
metacarpal elasticity. With low Ca and P levels in a 2 to 1 ratio,
Mn supplementation depressed rib Mn content, pancreas dry matter and
serum Mn, and increased metacarpal elasticity but did not change meta—
carpal Mg. With high Ca and P levels in a 2 to 1 ratio, Mn supplementa-
tion increased rib and serum Mn levels and pancreas dry matter, and
slightly increased metacarpal Mg but depressed metacarpal elasticity.
The deleterious effects on weight gain and feed efficiency of a low
dietary P level (0.35Z), derived exclusively from corn and soybean
meal, were much more pronounced than the effects of excessive dietary
levels of Ca and P or of an inverse Ca to P ratio, regardless of

dietary Mn concentration.

1. Histopathologyl

a. Histopathologic examination. The histology of the epiphyseal

cartilagenous plate was considered normal in all bones from all pigs

regardless of treatment group. There was some persistence of cartilage

.-—-—___—

1Performed by Dr. K. K. Keahey, Department of Pathology, Michigan
State University.

 

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Ho OVMN

 

 

 

 

 

 

 

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MN xH H0>0H also
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wMMngm
swapmsmz mzom zo Hm>mq zz oz< Hm>mH m 02¢ mo .oHamm misc zmmszm onHu<mmHzH mme mo Hommmm .NN mqmms

 

 

96
in the spicules. The most striking histologic change was in the diaphysis
of the bone. Pigs that had a thickness of compact bone equivalent to
70% of the diameter of the microscope field1 or greater had normal histo—
logic structures. Those with less than 70Z had incomplete ossification
of the Haversian canal system. The center of the Haversian canal was
occupied by a blood vessel which was surrounded by a significant zone
of loose connective tissue and osteoid. The compact bone, therefore,
appeared histologically like spongy bone instead of normal compact bone.
Examination of a record kept at the time of trimming of tissues indi—
cated that most of the soft bones had either a very thin layer of compact
bone in the diaphysis or an absence of compact bone. It was concluded
that the condition observed in the bones from pigs from some dietary
regimes were not changes of rickets but were changes in which there was
a failure of production of compact bone in the region of the diaphysis.
This conclusion agrees with that of Smith et al. (1944), who, using
X—ray pictures and microscopic studies, reported bone changes in Mn
deficiency were distinctly different from those seen in rickets. Neher
at al. (1956) also reported generalized bone rarefaction in Mn deficient

pigs.

b. Statistical inferences. Manganese supplementation was
associated with a significantly (P<0.05) higher histological normalcy
score of the epiphysis as compared to no Mn supplementation. The histo~
logy of the diaphysis was not affected by diet Mn level. Animals on
high Mn level diets had significantly (P<0.05) less compact bone in
the diaphysis, but these levels did not significantly affect the thick—

ness of the epiphyseal cartilagenous plate (Table 21). The ratio of

E...—

110X eyepiece and 20X objective.

 

 

 

 

 

97

Ca to P, level of Ca and P and the interaction between the two had a
significant effect (Table 23) on the bone histologic structure as
reported in many studies of Ca and P on bone metabolism (Underwood,
1971). The interaction between the Ca—P ratio and Mn levels was
significant (P<0.05) relative to the histologic structure of the
epiphysis (Table 25). With a 2 to 1 Ca to P ratio, Mn supplementation
increased the incidence of change in bone histologic structure. Mn
interaction with the Ca and P levels significantly (P<0.05) affected
the thickness of epiphyseal cartilagenous plate (Table 26). Produc-
tion of the normal histologic structure of the epiphyseal bone by Mn
supplementation is in agreement with the established concept that Mn
is involved in bone formation (Leach, 1967; Tsai and Everson, 1967).
However, the deleterious effects of a low dietary P level (0.35Z)
derived exclusively from corn and soybean meal on the histology of
bone were much more pronounced than the effects of high dietary levels
of Ca and P or of an inverse Ca to P ratio, regardless of dietary Mn
supplementation.
D. Experiment 4: The manganese requirement of the baby pig from sows

fed a low manganese diet

Table 28 summarizes the results of the analysis of variance.
Average daily gain was significantly (P<0.01) different between treat-
ments but feed efficiency was not. Absolute Mn intake, and absolute
fecal and urinary Mn excretion, were significantly (P<0.01 or P<0.05)
different between treatments, but absolute Mn retention was not. Mn
retention, fecal and urinary Mn excretion, all as percent of intake,
were significantly (P<0.01 or P<0.05) different between dietary treat—

ments. Of the serum parameters studied, serum Mg levels on the 28th day

 

 

.MM‘vA

 

 

 

 

98

TABLE 28. ANALYSIS OF VARIANCE OF EFFECT OF DIETARY MN LEVEL ON GROWTH,
BALANCE AND BLOOD MEASURES

 

 

P—level of F—
Itam Mean Min. Max. statistic
Serum parameters 1
Magnesium, mg/100 ml 9 2.38 2.00 2.85 0.83
21 2.57 1.87 3.05 0.49
28 2.04 1.26 2.90 0.03
Alkaline phosphatase
(SAP), Sigma units 9 5.8 3.4 8.4 0.04
21 7.0 5.3 10.3 0.10
28 6.2 3.0 10.7 0.11
Manganese,
mcg/lOO ml 9 1.20 .90 1.90 0.348
21 0.94 .60 1.50 0.129
28 0.90 .40 1.70 0.005
Growth data
Average daily gain, g 253 206 299 0.02
Feed/gain 1.33 1.18 1.45 0.238
Balance data
Mn intake, mg/day 1.82 0.18 4.06 <0.0005
Mn excretion, mg/day
Fecal 1.40 0.24 3.45 0.006
Urinary 0.015 0.01 0.02 0.049
Mn retention, mg/day .44 —.30 1.59 0.240
Mn retention, Z of intake 3.6 —72 73 0.016
Mn excretion, Z of intake
Fecal 94 26 167 0.02
Urinary 2.1 0.3 5.6 <0.0005

 

1Days on experimental diets.

 

 

 

 

 

99

of the experiment, serum alkaline phosphatase (SAP) on the 9th day and
serum Mn on the 28th day showed significant (P<0.01 or P<0.05) treat-
ment effects.

There were no treatment effects on serum Mg levels 9 days after
the start of the experiment, a slight rise on the 2.67 ppm Mn diet
(diet 2) after 21 days, and a significant (P<0.05) drop on the basal
diet by the end of the experiment (Table 29, Figure 4.1). Manganese
supplementation appeared to sustain normal serum Mg levels. The SAP
levels were high on the 6.34 ppm Mn diet (diet 3) and low on the basal
diet (0.46 ppm Mn) and diet 2 throughout the duration of the experiment.
0n the 9th and let days, the animals on the basal diet had higher
SAP levels than those on diet 2 and those values dropped slightly by the

end of the experiment (Table 29, Figure 4.2). The serum Mn values

 

were significantly (P<0.01) lower on the basal diet than on the diet
containing 6.34 ppm Mn on the 28th day of the experiment. There was
an appreciable and consistent drop of serum Mn values on the basal diet
and diet 2 as the experiment progressed. In animals on the 6.34 ppm
Mn diet, serum Mn values dropped slightly by the let day but returned
to previous levels by the 28th day (Table 29, Figure 4.3).

Serum Mn response to dietary Mn levels has been reported in swine
fed practical diets (Plumlee et al., 1956; Newland and Davis, 1961),
in ruminants (Hawkins at aZ., 1955; Rojas et aZ., 1965), and in chickens
(Bolton, 1955). A rise in SAP levels in response to Mn supplementation
has also been reported by some (Wachtel at aZ.. 1943; Lassiter gt aZ.,
1970) but not by others (Leibholz et al., 1962). The big drop of serum
Mg and serum Mn levels by the 28th day on the basal diet may indicate
that when animals are fed low Mn diets (0.46 ppm) for a long period

their Mn stores are depleted and Mg substitutes in the functions that

 

100

TABLE 29. THE EFFECT OF DIETARY MN LEVELS ON SERUM AND GROWTH PARAMETERS

 

 

 

 

Diets
Item 1 2 3
Mn conc., ppm 0.46 2.67 6.34
No. of pigs 4 4 4
i
[ Serum Mg, mg/100 ml 93 2.44 2.38 2.32
21 2.38 2.721/ 2.62
28 1.47 2.40— 2.25
SAP,4 Sigma units 9 5.6 4.6 7.3;]
21 7.0 5.8 8.3
28 4 5 5.5 8.6
Serum Mn, meg/100 ml 9 0.98 1.33 1.31
21 0.73 0.95 1.152/
28 0.41 0.88 l 28—
Average daily gain, g 224 283£/ 251
Feed/gain 1.37 1.27 1.35

 

lSignificantly (P<0.05) greater than the least value on the
same line.

2P<0.01.

3Days on experimental diets.

4Serum alkaline phosphatase.

 

 

that
high
sign
diet
rape:
in tl
suppc
ments
data
level
I
distal
the th
fecal
that o
statis
(P<0.01
abSOIut
reflect
rehone
s[JP-ties

the P111

 

 

 

 

101

usually involve Mn. Magnesium and Mn can replace each other in a number
of enzymatic reactions since they have similar properties (Lehninger,
1950, 1970). Johnson (1943) found a lower body Mn content of pigs born
of sows depleted of Mn. In this study, the correlations between serum
Mg and serum Mn were positive and significant (P<0.05) (Table 32).

Average daily gain was significantly (P<0.05) greater on diet 2
than on diet 1 (Table 29, Figure 4.4). The average daily gain was also
higher on diet 3 than on diet 1, but the difference was not statistically
significant. Food efficiency was greater on diet 2 than on the other
diets but those differences were not statistically significant. Many
reports have shown increased growth rate with optimal Mn supplementation
in the species studied (Underwood, 1971). Theresultsof this study
support those of Leibholz at al. (1962), who CODC1UdEd that Mn require—
ments for baby pig growth were very low. For maximal growth, these
data would indicate that 3.0 ppm Mn in the diet are adequate. This
level is approximately equal to that reported by Plumlee et al. (1956).

Mn intake, fecal Mn excretion and Mn retention all reflected the
dietary level of Mn (Table 30, Figure 4.5% The absolute Mn intakes of
the three diets were significantly (P<0.01) different; the absolute
fecal Mn excretion on diet 3 was significantly (P<0.01) greater than
that on diets l and 2 but the difference between diets 1 and 2 was not
statistically significant. Diets 2 and 3 resulted in significantly
(P<0.05) greater absolute urinary Mn excretion than diet 1 but the
absolute urinary Mn excretion on diets 2 and 3 was equal and did not
reflect the differing dietary Mn levels. Other workers have also
reported this small but virtually constant urinary Mn excretion in other
species irrepsective of Mn ingested and the dominance of the feces as

the principal excretion route of this element (Britton and Cotzias,

 

 

102

TABLE 30. RETENTION AND EXCRETION ROUTES 0F MN FROM DIETS CONTAINING
DIFFERENT LEVELS OF MN

 

 

 

 

Diets
Item 1 2 3
Mn conc., ppm 0.46 2.67 6.34
No. of pigs 4 4 4
Mn intake, mg/day 0.19 1.672! 3.632/5/
Mn excretion, mg/day 2/4/
Urinary .01 .02~ .02—
Mn retention, mg/day -.ll .60 .77
Mn retention, 2 of intake —58 36A! 22A/
Mn excretion, Z of intake 3/
Fecal 154— 4/ 63 78
Urinary 5.3“ 1.2 0.6

 

lSignificantly (P<0.05) greater than least value.

2P<0.01.

3Significantly (P<0.05) greater than least two values.

4P<0.01.

'49

im

 

 

 

 

103

1966; Miller, 1973). Absolute Mn retention did not differ signifi—
cantly between treatments. There was a negative absolute Mn retention
on the low dietary Mn level (-O.11 mg/day). This finding agrees with
the results of Zajcev (1959) and Starodubova (1968), who showed an
obligatory loss of Mn when animals were placed on very low Mn diets.
Mn retention, as a percent of intake, was significantly (P<0.05)
lower on the basal diet than on diets 2 and 3. Mn retention as a
percent of intake on diets 2 and 3 did not differ significantly. Hill
and Holtkamp (1954) reported a greater net absorption at a low Mn
concentration in the diet than at high concentrations, and Gutowska
et al. (1941) showed that net Mn absorption was proportional to dietary
Mn concentration. Fecal and urinary Mn excretion, as percent of intake,
were significantly (P<0.01 or P<0.05) higher on the basal diet than
on diets 2 and 3. The fecal and urinary Mn excretion, as percent of
intake, on diets 2 and 3 were not significantly different.

Dietary Mn was significantly (P<0.01) related to levels of serum
Mn on the 9th day, SAP on the 28th day and serum Mg on the 28th day of
the experiment (Table 31). Mn intake was significantly (P<0.01)
correlated to serum Mn on the 9th day, serum Mn on the 28th day, and
SAP on the 28th day of the experiment. Krieg (1966) found no parallel
between Mn in the diet and that in blood. There was a poor positive
correlation between Mn intake and average daily gain, and feed efficiency.
Average daily gain and feed efficiency were poorly correlated to all
the serum parameters except SAP on the 9th day. Serum Mg on the 28th
day was related to feed, urine and tissue Mn concentration. Absolute
fecal Mn excretion had a significant (P<0.01) positive correlation with
serum Mn on the 9th day and SAP on the 28th day of the experiment. Mn

retention as percent of intake was significantly (P<0.01 or P<0.05)

 

v ‘-ifih , (44

 

 

 

 

 

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104

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105
positively related to serum Mn throughout the experiment, serum Mg on
the 28th day and average daily gain. This may indicate that a high
retention of Mn enhances growth rate. Fecal and urinary Mn excretion,
both as a percent of intake, were significantly (P<0.01 or P<0.05)
negatively correlated to serum Mn throughout the experiment, and serum
Mg on the 28th day. Feed efficiency was significantly (P<0.05)
negatively related to Mn retention as percent of intake. Mn retention
and fecal Mn excretion, both as percent of intake, had high correla—
tions with average daily gain and were better indicators of growth rate
than absolute Mn intake, excretion or retention, or any of the serum
parameters studied. Up to the let day of the experiment, none of the
serum parameters were significantly correlated with each other. On

the 28th day, serum Mn had significant (P<0.05) positive correlations

 

with serum Mg and SAP (Table 32).

Correlations within the balance measures are summarized in Table

 

33. Dietary Mn level was positively correlated to Mn intake, excretion
and Mn retention as percent of intake. However, these correlations
were significant (P<0.01) only with Mn intake and absolute fecal Mn
excretion. Urinary Mn excretion as a percent of intake was signifi—
cantly (P<0.01) negatively correlated to dietary Mn levels. Woerpel
and Balloun (1964) and Hill and Holtkamp (1954) reported that retention
was highly related to dietary Mn levels. Mn intake and absolute fecal
and urinary Mn excretion were all positively correlated to Mn retention
as percent of intake and absolute Mn retention. Mn intake was sig~
nificantly (P<0.01) positively correlated to urinary Mn excretion as
Percent of intake. Absolute Mn retention had high correlations with
Mn retention as percent of intake (r = + 0.85) and fecal Mn excretion

as percent of intake (r = ~ .85). There was a poor correlation between

 

IE!

 

106

TABLE 32. CORRELATIONS WITHIN THE SERUM PARAMETERS

 

 

Periodl Comparison2 r3
9th day SMn vs SAP 0.31
SMn vs SMg -.l3
SMg vs SAP 0.09
let day SMn vs SAP 0.31
SMn vs SMg 0.08
SMg vs SAP 0.03
28th day SMn vs SAP 0.57%;
SMn vs SMg 0.60—
SMg vs SAP 0. 39

 

 

 

 

 

1Days after the start of the experiment.

2SMn, SMg, SAP (Serum Mn, Mg and alkaline phosphatase,
respectively).

3Correlation coefficient.

4Significant (P<0.05).

 

 

‘

._-
2"”.

Mil

REPI
FEPI

LTPI

absol-
h- re-
Of inI

 

 

 

107

TABLE 33. CORRELATIONS WITHIN THE MN BALANCE MEASURESl

 

Feed Mn
Mn Intake FE UE RET REPI FEPI UEPI

 

Feed Mn 1.00 .993/ .873/ .52 .50 .54 -.52 4341/
Mn intake 1.00 .88g/ .54 .50 .56 -.54 -.862/
FE 1.00 .45 .03 .19 —.17 —.67—3-/
UE 1.00 .31 .51 -.50 —.633/
RE 1.00 +. 853/ -. 853/ —. 603/
REPI 1.00 -1.oo§/ —.8o§/
FEPI 1.00 .793/
UEPI ~ 1.00

 

 

lAbbreviations are as follows: FE = fecal Mn excretion; UE =
absolute urinary Mn excretion; RE = absolute Mn retention; REPI =
Mn retention as percent of intake; FEPI = fecal Mn excretion as percent
of intake; UEPI = urinary Mn excretion as percent of intake.

2Significant at P<0.01.

3Significant at P<0.05.

abs
Sn

and

int:

EXCI

351’

In t

hniI

intal

hathe

 

 

108
absolute fecal Mn excretion and absolute Mn retention. Mahoney and
Small (1968) reported a good relationship between fecal Mn excretion
and body retention. Mn retention as percent of intake had a perfect
(r = - 1.0) negative correlation with fecal Mn excretion as percent of
intake and a highly significant (P<0.01) correlation with urinary Mn
excretion as percent of intake. Urinary and fecal Mn excretion, both
as percent of intake, were significantly (P<0.01) positively related.
In this study, there was no significant positive correlation between
Mn intake and absolute Mn retention or Mn retention as percent of
intake, which is contrary to Murty's (1957) findings in sheep and to

Mathers' and Hill‘s (1967) findings in chickens.

 

109

Figure 4.1. The effect of dietary Mn concentration on serum Mg
concentration.

 

110

A52 Eqavmbv m ._.m:Q Qilo

 

.H.q wudwfim

£65 EmECoaxm co 980
mm _N

 

O._

 

O.m

IwOOI/bw ‘5W ”W93

 

111

Figure 4.2. The effect of dietary Mn concentration on serum
alkaline phosphatase activity.

._/

SSDJ

 

112

N

.q wuswflm

22v BEmEtmaxm co mzoo

mN _N m.
. _

O.N

32 533.8 m 55 o--lo
2.2 can 5.8 N Ba 9:6 -
Ass. EQQQVOV _._.m_DOI-I.O O\

 

I00.

 

 

suun owbgg

asmnudsoqd auumuv wmag

Figure 4.3.
concentration.

113

The effect of dietary Mn concentration on serum Mn

 

(O. 46 ppm Mn)

o—--o DIET 2 (2.67 ppm Mn)
o—-o DIET 3 (6.34ppm Mn)

0——0 DIET I

1.5:-

.m.e nnnwsm
965 _2cmE_._maxw :0 960
mm mm _N t m. m

—

 

114

 

- m._
22 £3va m Ea ¢-|o
Es. 58 5.8 m Ea 9:6
22 588.2 _ emaollo

 

[mom/50w ‘UW wmeS

115

Figure 4.4. The effect of dietary Mn concentration on average
daily gain (ADC) and feed efficiency (F/G).

 

 

12'!

9'28?

 

 

 

 

III-IIII ' — All
. IIIIIIIIIIIIIII '

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(we) qu9 KIIDO 95mm

DIET 3
6.34

DIET 2
2.67

E
4.4.

I: A D G
Fig...

 

 

 

 

117

Figure 4.5. Mn balance data, intake, fecal and urinary excre—
tion and retention on different dietary Mn levels.

4.0

3.0

anxanuslarwensez (anggz1bczy)

2.0 -

 

DMn
EMn
.Mnl

DIET I
(0.46 ppm

 

118

4.0

 

A [3 Mn intake
EMn Fecal Excretion
’5, - Mn Urinary Excretion
O
\ 3.0—
at
.2.
LIJ
(I)
LIJ
<2): /
0 2.0 -
i Z
I <1
I 2 7
IO -

 

 

 

o E
DIET | DIET 2
(0.46 ppm Mn) (2.67 ppm Mn)

Figure 4.5.

 

 

 

 

 

 

DIET 3
(6.34 ppm Mn)

 

 

 

Within the limits
employed, the results
1. Growth rates
com-soy basal diet or
amounts of Mn from eit?
2. Mn from the 5-
young pig than that fr-
and tissue Mn concentra
3. Regardless of
was recovered in the fa
was minimal and constar.
very low dietary Mn lex-
excretion resulting in
1i. The requiremen
was 3.0 ppm Mn in the d
for maintenance of norm
min-um gain over longe
5. Serum Mn and s-
correlated with Mn intaI
were not. Mn retention
had high correlations w:

of growth rate than abs-

Iln serum parameters .

 

CONCLUSIONS

Within the limits of the experimental conditions and procedures
employed, the results of this study lead to the following conclusions:
1. Growth rates were equal whether Mn was derived from the 16%
corn—soy basal diet or from the same diet supplemented with equal
amounts of Mn from either the sulfate or the carbonate or the oxide.
2. Mn from the supplemented diets was more available to the
young pig than that from corn and soy alone when assessed by Mn balance

and tissue Mn concentrations.

3. Regardless of dietary Mn source, over 90% of the excreted Mn

was recovered in the feces; absolute excretion of Mn via the kidneys
was minimal and constant irrespective of dietary Mn levels; and on
very low dietary Mn levels ( 0.50 ppm), there was an obligatory Mn

excretion resulting in a negative Mn retention.

4. The requirement for maximal daily gain over a 4-week period

was 3.0 ppm Mn in the diet of baby pigs but may be higher (3—6 ppm)
for maintenance of normal serum Mn concentration (and possibly for
maximum gain over longer periods).

5. Serum Mn and serum alkaline phosphatase levels were highly

correlated with Mn intake, but average daily gain and feed efficiency
were not. Mn retention and fecal Mn excretion, as percent of intake,
had high correlations with average daily gain and were better indicators
of growth rate than absolute Mn intake, excretion and retention, and

the serum parameters.

119

 

6. Mn from the a
stomach, cranial small
soy was apparently abs
absorption of Mn from
supplemented diets, at
intestine on the supp]

7. Mn supplement
affect hemoglobin and
Plevels, bone physics
parameters.

8. Although Mn 5
structure of the epipt
in the region of the c

9. There was a a
and Mn levels in the a
structure that were d:

It should be empl
gain, feed efficiency
level (0.35%) from so:
effects of excessive a

ratio, regardless of 1

 

120

6. Mn from the supplemented diets was apparently absorbed in the
stomach, cranial small intestine and cecum, whereas Mn from corn and
soy was apparently absorbed in the stomach and cecum only. Net cecal
absorption of Mn from the basal diet was higher than that of Mn from
supplemented diets, and net secretion of Mn into the caudal small
intestine on the supplemented diets was higher than on the basal diet.

7. Mn supplementation of a 16 ppm Mn diet did not significantly
affect hemoglobin and hematocrit values, serum Ca, Mg, and inorganic
P levels, bone physical measurements, breaking strength or related
parameters.

8. Although Mn supplementation produced a more normal histologic
structure of the epiphysis, it reduced the thickness of compact bone
in the region of the diaphysis.

9. There was a significant metabolic interaction between Ca, F
and Mn levels in the diets of pigs, which produced changes in bone
structure that were distinctly different from those seen in rickets.

It should be emphasized that the deleterious effects on weight
gain, feed efficiency and the histology of bone of a low dietary P

level (0.35%) from soybean meal were much more pronounced than the
effects of excessive dietary levels of Ca and P or an inverse Ca to P

ratio, regardless of dietary Mn supplementation.

 

 

 

 

 

 

 

 

 

Amdur, B. H., H. Ril]
of mevalonic a

Amdur, M. 0., L. C. N
action of mang

Anderson, B. M. and H
manganese and
concentration

Angelico, R.,L. Maggi
ents in the di
and nitrogen at
lst Super. San

Angelica, R.,M. Manga1
Influence of i]
of ionizing rac
Super. Sanita,

Anke, M., H. Joroch, l
and distributic
ganese salts i!

Anke, M.and B. Groppel
studies on mang
Element Metab.
Edinburgh.

Anong—Nilubol, M. L.,
Manganese toxic
analysis. J. 1\‘

Antanova, M. V. 1968.
on immunologica

Antanova, M. V., V. A.
small amounts 0
responses. Gig

Archibald, J. G. and J
J. Dairy Sci. 2

Atkinson, R. L., J. W.
Effect of vario
turkeys. Poul.

 

 

 

 

 

#7— ' ' fi'flwwmmmmmmm""

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‘llllllllllIl-IIL_ - ______aiil__11=wrfifi=fififw__rr____,,7
'i
l

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1F—_————

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Radoin, In 1944' (
iron, mangam
of all these
diets contair

Ray, T- W- and L' ‘1'
Effect on 0X?
Biol. Med. 51

Rehner, G. and W. St
nickel on liv
Ernahrung. ll

Rehner, G. and H. D.
ment by estim

Reineke, E. P. and C
pounds and ce:
in iodinated r

Richards, M. B. 193(
J. 24: 1572.

Robinson, N. W., S. I
Excess dietary
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Rojas. M. A., I. A. n
in the bovine.

Rothstein, A., A. Hay
active transpo
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Rozybakiev, M. A. 19‘
sheep. Trudy‘

Rubenstein, A. An N-
induced by man:

WIN. N. 1939.
ascorbic acid.

I

Rudra, M. N. 1944a.

MW- N- 1944b.
ascorbic acid.

Sahagian, 3' M-. I Ha

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Sahagian, B. M I Ha

. " s .
anOlvements of
lntEStine. J. 1

 

 

134

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5%

 

 

Saltman. Pu R- D. Fi
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Supplements on

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Effect of diet
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toms 0f mangan

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otoliths assoc
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Shrader, R. E. and G
In"'mganeSe defi

Sigma Technical Bulle
alkaline and 1)
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r________fl

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—_¥

 

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Cobalt and mag.
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i 4 77m;

x
1;

 

 

 

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E. I

Ullrey 1" E"
’Hoeffer- 19
Serum calcil
inorganic pl

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3rd edition'

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manganese fC

Van Reen, 1" and P.
duck. J- Nu

Vohra. P- and J' R'
manganese an

Vorob'eva, A' M’ K'
ganese on an

Wachtel, 1“ W., 0'
phy51010gy 0
72.

Wacker, W. E. C. any
IIA (c. L- 0‘

Watson, L. T., (3' B
Influence of
other minera.

Watson, L. Tu C° 3‘
Biological a:
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Watson, L. T., C- B.
Biological ax
inorganic sot

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as a means of

rung. 10: 142

wWinger, o. D.

and
boron comp

0111'!

 

 

137

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C.
Wiese A- C., 3'

’Halpin. 193'

J. Biol. CheI

Wilgus, H. 3., L- C
certain inor;
perosis. Sc:

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C01“Pounds by

Zerebeov, P, 1., V.
trace element:
metabolism in

' 159.

 

F.-- 77* i if i W

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4: 159.

 

 

 

 

 

 

i APPEND IX

 

 

 

 

 

 

 

 

APPENDIX A

 

 

 

TABLE A-l. MICHII

___.——

Nutrient

—..

Vitamin A, 111:
Vitamin D2, 11
Vitamin E, t}
Riboflavin
Nicotinic aci
Choline chlor
D-pantothenic
Vitamin B12
Zinc
Manganese
Iodine

Copper

Iron
Antioxidant

Carrier (ground ye.

_\

1
Ten pounds

2
Butylated 1

TABLE A-2. MINERAI

KN
Nutrient

\

KCl

KI
FeSO4.2H20
CuSO4
COC03
MnSO4.H20
ZnSO
MgHCO3
CaHP04.2H20
CaCO3
cerelose

...J¥§E§§L_‘_‘_‘_‘_
l 2
’ For 3 and

 

TABLE A-l. MICHIGAN STATE UNIVERSITY VITAMIN-TRACE MINERAL PREMIX

 

Nutrient AmOunt in 10 lbs of premixl

 

Vitamin A, million 3.0 I.U
Vitamin D2, million 0.6 I.U
Vitamin E, thousand 10.0 I.U
Riboflavin 3.0 gm
Nicotinic acid 16.0 gm
Choline chloride 100.0 gm
D—pantothenic acid 12.0 gm
Vitamin B12 18.0 gm
Zinc 68.0 gm
Manganese 34.0 gm
Iodine 2.5 gm
Copper 9-0 8m
Iron 2 54-0 gm
Antioxidant 45-0 gm

Carrier (ground yellow corn) to bring total to 10 lbs

 

lTen pounds of premix mixed in 1 ton of feed.

2Butylated hydroxyanisole (BHA) and/or butylated hydroxytoluene (BHT).

TABLE A-2. MINERAL MIXTURE USED IN SEMI-PURIFIED EXPERIMENTAL DIETS

 

Nutrient Percentage in diet

 

O

KCl

KI
FeSO402H20
CuSO4
C0CO3
MnSO4'H20
ZnSO4
MgHCO3
CaHPO4°2H20 12.5 1/

caCO3 13.098, 13.089—
Cerelose 13 071 2/
.o —

OO
O
N

.009;/, .0272]

NOOOOOOH
ObOI-‘I—‘NO'

b)
o. .
O

NaHCO3 25

l’2For 3 and 9 ppm Mn supplementation of the diets 2 and 3.

139

nmLEA-3. VITAMI

#

Nutrient

Thiamine monon
Riboflavin
Nicotinamide
Calcium pantot
Pyridoxine HCl
Para-amino ben:
Ascorbic acid
D-a-tocopheryl
Inositol
Choline chloric‘

Pteroyl glutami
Biotin
Cyanocobalamin
2-methyl—1, 4-“,
Vitamin A Palmi1
Vitamin D2

\
TABLE A-4. CLEANING

N

Polyethylene Via;
ing SOlutions;

1' XYlene

2' 9570 6 th)
3' Dis tille
4' (0.1N) d
5- Distille

The vials Were:

1' Dried in
2‘ Numbered

\

 

140

TABLE A-3. VITAMIN MIXTURE USED IN SEMI-PURIFIED EXPERIMENTAL DIETS

 

Nutrient
______________.__________________________________________________________
ppm in diet
Thiamine mononitrate 3
Riboflavin 6
Nicotinamide 40
Calcium pantothenate 30
Pyridoxine HCl 2
Para-amino benzoic acid 13
Ascorbic acid 80
D—a—tocopheryl acetate 10
Inositol 130
Choline chloride 1300
ppb in diet
Pteroyl glutamic acid 260
Biotin 50
Cyanocobalamin 100
2—methyl—l, 4—naphthoquinone 40
Vitamin A palmitate 1500
Vitamin D2 12.5

 

TABLE A—4. CLEANING POLYETHYLENE VIALS

 

Polyethylene vials were cleaned successively with the follow—
ing solutions:

Xylene

95% ethyl alcohol
Distilled deionized water
(0.1N) dilute nitric acid
Distilled deionized water

LJ'I-L‘LIJNH

The vials were:

1. Dried in a draft oven at 100°C immediately prior
to loading

2. Numbered by engraving letters using a stainless
metal rod

TABLE A—5. IL MOD]

1. Instrument
MOde of
Hollow c
Lamp cu]
Photomul
P.M. vol
Slit wit
waveleng

2. Recorder <
Response
Range

3. Parameters
Mode
Purge ga
Gas flow
Dry sett
Pyrolyze

Height 0
Analyse

\\

 

141

TABLE ArS. IL MODEL 335 FLAMELESS SAMPLER

 

1. Instrumental parameters

Mode of operation AeB Channel B
Hollow cathode 45472 Hz continuum
Lamp current SmA #IL 63490
Photomultiplier R456 20mA
P.M. voltage 620V
Slit width 320 um
wavelength 279.5
2. Recorder (full scale)
Response time 0.5 sec
Range 100 mV
3. Parameters for 355
Mode Manual
Purge gas Argon
Gas flow rate 10 SCFH
Dry setting 4 turns
Pyrolyze setting 5 turns on
Analyse Scale
Height of measurement 2 mm

Analyse setting 10 turns

 

 

 

 

 

‘.
.
4.
,
I
A