Tissue copper accumulation as a measure of bioavailability in chicks and sheep

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Tissue copper accumulation as a measure of bioavailability in chicks and sheep
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Ledoux, David Randolph, 1952-
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Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
        Page iv
    Table of Contents
        Page v
        Page vi
        Page vii
    List of Tables
        Page viii
        Page ix
        Page x
        Page xi
    List of Figures
        Page xii
    Abstract
        Page xiii
        Page xiv
    Chapter 1. Introduction
        Page 1
        Page 2
        Page 3
    Chapter 2. Review of literature
        Page 4
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    Chapter 3. Effect of high dietary copper on tissue mineral composition of broiler chicks as a bioassay of copper sources--Experiment 1
        Page 44
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    Chapter 4. Effect of high dietary copper and age on tissue mineral composition of broiler chicks as a bioassay of copper sources--Experiment 2
        Page 59
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    Chapter 5. Biological availability of copper from inorganic sources fed at high dietary levels to broiler chicks--Experiment 3
        Page 83
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    Chapter 6. Effect of high dietary copper and time on tissue mineral composition of sheep as a bioassay of copper sources--Experiment 4
        Page 114
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    Chapter 7. Effect of time and high dietary copper on tissue mineral composition of sheep depleted of copper for three weeks--Experiment 5
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    Chapter 8. Biological availability of copper from inorganic sources fed at high dietary levels to sheep depleted of copper for three weeks--Experiment 6
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    Chapter 9. General summary and conclusions
        Page 188
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    Literature cited
        Page 194
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    Biographical sketch
        Page 219
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Full Text












TISSUE COPPER ACCUMULATION AS A MEASURE
OF BIOAVAILABILITY IN CHICKS AND SHEEP


















BY

DAVID RANDOLPH LEDOUX


















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



UNIVERSITY OF FLORIDA

1987


































TO

ZENA,

RICHARD

AND

SHANE















ACKNOWLEDGEMENTS


The author wishes to express his heart-felt appreciation

to Dr. Clarence B. Ammerman, chairman of the supervisory

committee, for his guidance, encouragement and understanding

during this period of study. Sincere appreciation is also

extended to Drs. John E. Moore, Richard D. Miles, Pejaver V.

Rao and 0. Charles Ruelke, members of the supervisory

committee, for giving freely of their time and knowledge.

Appreciation is extended to Dr. H. N. Becker for performing

biopsies and to Dr. Roderick McDavis for his support and

assistance.

The author is especially indebted to Mrs. Pamela H.

Miles for her assistance and suggestions in all phases of

the experiments and for reviewing the first draft of the

manuscript. Special thanks is extended to Ricardo van

Ravenswaay, Francis Tarla, Mariano Echevarria, Jose

Wong-Valle, Gary Russell, Jack Stokes and others who

assisted in necropsy of animals. The friendship and support

of the nutrition laboratory staff and fellow graduate

students are also greatly appreciated.

Acknowledgement is made to Moorman Manufacturing

Company, Quincy, Illinois; Pitman-Moore, Inc., Mundelin,

Illinois; and Occidental Chemical Agricultural Products,


iii









Inc., Tampa, Florida, for financial support of this

research. The provision of experimental supplies by

Monsanto Chemical Company, St. Louis, Missouri; Pfizer,

Inc., New York, New York; and Southeastern Minerals,

Bainbridge, Georgia, is gratefully acknowledged.

Acknowledgement is also made to International Minerals and

Chemical Corporation, Fertilizer Group, Noralyn, Bartow,

Florida, for X-ray diffraction analysis and to Southeastern

Minerals, Inc., Bainbridge, Georgia, for having selected the

author to be a recipient of BASF Corporation's "Growth is a

Promise" scholarship grant.

Sincere appreciation is expressed to Mrs. Janet Eldred

for typing this dissertation.

The author expresses special thanks to his mother, Joan,

for her love and support and to his wife, Zena, for her

understanding, inspiration, forbearance and love.

Finally, I would like to dedicate this manuscript to my

sons, Richard and Shane, that it might serve as an example

of what can be accomplished through hard work and

dedication.















TABLE OF CONTENTS


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

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

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

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

CHAPTERS

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

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

Copper Metabolism .............................

Absorption ...............................
Transport ................................
Excretion ................................

Copper Deficiency and Functions ...............

Anemia and Iron Metabolism ...............
Pigmentation and Keratinization ..........
Skeletal Abnormalities ...................
Cardiovascular System ....................
Central Nervous System ...................
Reproduction .............................

Copper Toxicosis ..............................
Copper Bioavailability ........................

III EFFECT OF HIGH DIETARY COPPER ON TISSUE
MINERAL COMPOSITION OF BROILER CHICKS
AS A BIOASSAY OF COPPER SOURCES--
EXPERIMENT i ..................................

Introduction ..................................
Materials and Methods .........................
Results .......................................
Discussion ....................................
Summary .......................................


Page

iii

viii

xii

xiii



1

4

5

5
7
9

10

11
14
15
17
19
25

29
37




44

44
45
48
52
57









Page

IV EFFECT OF HIGH DIETARY COPPER AND AGE ON
TISSUE MINERAL COMPOSITION OF BROILER
CHICKS AS A BIOASSAY OF COPPER SOURCES--
EXPERIMENT 2 .................................. 59

Introduction .................................. 59
Materials and Methods ......................... 60
Results ....................................... 63
Discussion .................................... 78
Summary ....................................... 81

V BIOLOGICAL AVAILABILITY OF COPPER FROM
INORGANIC SOURCES FED AT HIGH DIETARY
LEVELS TO BROILER CHICKS--EXPERIMENT 3 .......... 83

Introduction .................................. 83
Materials and Methods ......................... 84
Results ....................................... 89
Discussion .................................... 106
Summary ....................................... 112

VI EFFECT OF HIGH DIETARY COPPER AND TIME ON
TISSUE MINERAL COMPOSITION OF SHEEP AS A
BIOASSAY OF COPPER SOURCES--EXPERIMENT 4 ........ 114

Introduction .................................. 114
Materials and Methods ......................... 115
Results ....................................... 118
Discussion .................................... 131
Summary ....................................... 135

VII EFFECT OF TIME AND HIGH DIETARY COPPER
ON TISSUE MINERAL COMPOSITION OF SHEEP
DEPLETED OF COPPER FOR THREE WEEKS--
EXPERIMENT 5 .................................. 137

Introduction .................................. 137
Materials and Methods ......................... 139
Results ....................................... 142
Discussion .................................... 155
Summary ....................................... 163

VIII BIOLOGICAL AVAILABILITY OF COPPER FROM
INORGANIC SOURCES FED AT HIGH DIETARY
LEVELS TO SHEEP DEPLETED OF COPPER FOR
THREE WEEKS--EXPERIMENT 6 ..................... 166

Introduction .................................. 166
Materials and Methods ......................... 167
Results and Discussion ........................ 172
Summary ........................................ 185









Page

IX GENERAL SUMMARY AND CONCLUSIONS ............... 188


LITERATURE CITED ...................................... 194

BIOGRAPHICAL SKETCH ................................... 219


vii















LIST OF TABLES


Table Page

3-1 COMPOSITION OF BASAL DIET--EXPERIMENT 1 ........ 46

3-2 EFFECT OF DIETARY COPPER CONCENTRATION
ON FEED INTAKE, GAIN AND FEED CONVERSION
IN CHICKS--EXPERIMENT 1 ....................... 49

3-3 EFFECT OF DIETARY COPPER CONCENTRATION
ON MINERAL COMPOSITION OF LIVER AND
KIDNEY IN CHICKS--EXPERIMENT 1 ................... 50

3-4 EFFECT OF DIETARY COPPER CONCENTRATION
ON BONE ASH PERCENTAGE AND MINERAL
COMPOSITION OF BONE AND PLASMA IN
CHICKS--EXPERIMENT 1 .......................... 53

4-1 COMPOSITION OF BASAL DIET--EXPERIMENT 2 ........ 61

4-2 EFFECT OF DIETARY COPPER AND AGE ON FEED
INTAKE, GAIN AND FEED CONVERSION IN
CHICKS--EXPERIMENT 2 .......................... 64

4-3 EFFECT OF DIETARY COPPER AND AGE ON THE
MINERAL COMPOSITION OF LIVER IN CHICKS--
EXPERIMENT 2 .................................. 65

4-4 EFFECT OF DIETARY COPPER AND AGE ON THE
MINERAL COMPOSITION OF KIDNEY IN CHICKS--
EXPERIMENT 2 .................................. 69

4-5 EFFECT OF DIETARY COPPER AND AGE ON THE
MINERAL COMPOSITION OF MUSCLE IN CHICKS--
EXPERIMENT 2 .................................. 71

4-6 EFFECT OF DIETARY COPPER AND AGE ON BONE
ASH PERCENTAGE AND MINERAL COMPOSITION
IN CHICKS--EXPERIMENT 2 ....................... 74

4-7 EFFECT OF DIETARY COPPER AND AGE ON THE
MINERAL COMPOSITION OF PLASMA IN CHICKS--
EXPERIMENT 2 .................................. 76


viii









Table Page

5-1 COMPOSITION OF BASAL DIET--EXPERIMENT 3 ....... 85

5-2 CHEMICAL AND PHYSICAL CHARACTERISTICS OF
COPPER SOURCES--EXPERIMENT 3 ..................... 87

5-3 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON FEED INTAKE, GAIN AND FEED
CONVERSION IN CHICKS--EXPERIMENT 3 .............. 91

5-4 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON MINERAL COMPOSITION OF LIVER
IN CHICKS--EXPERIMENT 3 ....................... 93

5-5 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON MINERAL COMPOSITION OF KIDNEY
IN CHICKS--EXPERIMENT 3 ....................... 98

5-6 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON BONE ASH PERCENTAGE AND
BONE MINERAL COMPOSITION IN CHICKS--
EXPERIMENT 3 .................................. 101

5-7 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON MINERAL COMPOSITION OF PLASMA
IN CHICKS--EXPERIMENT 3 ....................... 104

5-8 RELATIVE BIOLOGICAL AVAILABILITY OF
COPPER SOURCES BASED ON LINEAR REGRES-
SION AND LIVER COPPER CONCENTRATION--
EXPERIMENT 3 .................................. 107

6-1 COMPOSITION OF BASAL DIET--EXPERIMENT 4 ......... 116

6-2 EFFECT OF DIETARY COPPER LEVEL AND TIME
ON HEMOGLOBIN (Hb), HEMATOCRIT (Hct) AND
MINERAL COMPOSITION OF SERUM IN SHEEP--
EXPERIMENT 4 .................................. 119

6-3 EFFECT OF DIETARY COPPER LEVEL AND TIME
ON THE MINERAL COMPOSITION OF LIVER IN
SHEEP--EXPERIMENT 4 ........................... 122

6-4 EFFECT OF DIETARY COPPER LEVEL AND TIME
ON THE MINERAL COMPOSITION OF SPLEEN IN
SHEEP--EXPERIMENT 4 ........................... 125

6-5 EFFECT OF DIETARY COPPER LEVEL AND TIME
ON THE MINERAL COMPOSITION OF KIDNEY IN
SHEEP--EXPERIMENT 4 ........................... 127









Table Page

6-6 EFFECT OF DIETARY COPPER LEVEL AND TIME
ON BONE ASH PERCENTAGE AND MINERAL COMPO-
SITION OF BONE IN SHEEP--EXPERIMENT 4 ............ 129

7-1 COMPOSITION OF BASAL DIET--EXPERIMENT 5 ......... 140

7-2 EFFECT OF TIME AND DIETARY COPPER LEVEL
ON HEMOGLOBIN (Hb), HEMATOCRIT (Hct) AND
MINERAL COMPOSITION OF SERUM IN SHEEP--
EXPERIMENT 5 .................................. 144

7-3 EFFECT OF TIME AND DIETARY COPPER LEVEL
ON THE MINERAL COMPOSITION OF LIVER IN
SHEEP--EXPERIMENT 5 ........................... 146

7-4 EFFECT OF TIME AND DIETARY COPPER LEVEL
ON THE MINERAL COMPOSITION OF SPLEEN IN
SHEEP--EXPERIMENT 5 ........................... 151

7-5 EFFECT OF TIME AND DIETARY COPPER LEVEL
ON THE MINERAL COMPOSITION OF KIDNEY IN
SHEEP--EXPERIMENT 5 ........................... 153

7-6 EFFECT OF TIME AND DIETARY COPPER LEVEL
ON BONE ASH PERCENTAGE AND MINERAL COMPO-
SITION OF BONE IN SHEEP--EXPERIMENT 5 ............ 156

8-1 COMPOSITION OF BASAL DIET--EXPERIMENT 6 ......... 168

8-2 CHEMICAL AND PHYSICAL CHARACTERISTICS OF
COPPER SOURCES--EXPERIMENT 6 ..................... 171

8-3 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON HEMOGLOBIN AND HEMATOCRIT
IN SHEEP--EXPERIMENT 6 ........................ 175

8-4 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON THE MINERAL COMPOSITION OF
SERUM IN SHEEP--EXPERIMENT 6 ..................... 176

8-5 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON THE MINERAL COMPOSITION OF
LIVER IN SHEEP--EXPERIMENT 6 ..................... 178

8-6 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON LIVER COPPER ACCUMULATION
IN SHEEP--EXPERIMENT 6 ........................ 179

8-7 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON THE MINERAL COMPOSITION OF
SPLEEN IN SHEEP--EXPERIMENT 6 ................. 181









Table Page

8-8 EFFECT OF SOURCE AND LEVEL OF DIETARY
COPPER ON THE MINERAL COMPOSITION OF
KIDNEY IN SHEEP--EXPERIMENT 6 .................... 182

8-9 EFFECT OF SOURCE AND LEVEL OF
DIETARY COPPER ON BONE ASH PERCENTAGE
AND MINERAL COMPOSITION OF BONE IN
SHEEP--EXPERIMENT 6 ........................... 184
















LIST OF FIGURES


Figure Page

3-1 MEASURED AND PREDICTED COPPER CONCENTRA-
TIONS OF LIVER WITH RESPECT TO DIETARY
COPPER INTAKE IN CHICKS--EXPERIMENT 1 ........... 51

3-2 MEASURED AND PREDICTED COPPER CONCENTRA-
TIONS OF PLASMA WITH RESPECT TO DIETARY
COPPER INTAKE IN CHICKS--EXPERIMENT 1 ........... 54

4-1 MEASURED AND PREDICTED COPPER CONCENTRA-
TIONS OF LIVER WITH RESPECT TO DIETARY
COPPER INTAKE IN CHICKS FED DIETS FOR
2 OR 3 WEEKS--EXPERIMENT 2...................... 67

5-1 PREDICTED COPPER CONCENTRATIONS OF LIVER
WITH RESPECT TO DIETARY COPPER INTAKE IN
CHICKS--EXPERIMENT 3 .......................... 96

7-1 MEASURED AND PREDICTED COPPER CONCENTRA-
TIONS OF LIVER WITH RESPECT TO DIETARY
COPPER INTAKE IN SHEEP FED DIETS FOR
10 DAYS--EXPERIMENT 5 ......................... 149

7-2 MEASURED AND PREDICTED COPPER CONCENTRA-
TIONS OF LIVER WITH RESPECT TO DIETARY
COPPER INTAKE IN SHEEP FED DIETS FOR
20 DAYS--EXPERIMENT 5 ......................... 150















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


TISSUE COPPER ACCUMULATION AS A MEASURE

OF BIOAVAILABILITY IN CHICKS AND SHEEP

by

David Randolph Ledoux

December 1987

Chairman: Clarence B. Ammerman
Major Department: Animal Science

Six experiments were conducted to investigate the use

of tissue Cu accumulation during short term, high level Cu

supplementation as bioassay criterion to determine Cu avail-

ability from inorganic sources fed to chicks and sheep. In

Experiment 1, chicks were fed diets supplemented with up to

300 mg/kg Cu from reagent grade (RG) cupric acetate for 22

days to determine the relationship between dietary Cu and

tissue Cu. Copper concentrations in plasma and liver

increased with increasing dietary Cu. In Experiment 2,

chicks were fed the same levels and source of Cu as in

Experiment 1 for 1, 2 or 3 weeks to determine the effect of

age on tissue Cu accumulation. Liver was the only tissue

sensitive to Cu at weeks 2 and 3. Experiment 3 was a 3-week

bioassay conducted to determine Cu availability from RG

acetate, feed grade (FG) oxide, FG sulfate and FG carbonate.


xiii









Acetate and sulfate were equally available and were both

more available than carbonate which, in turn, was more

available than oxide. The relationship between dietary Cu

and tissue Cu in sheep fed up to 45 mg/kg added Cu from RG

acetate for 15 or 30 days was investigated in Experiment 4.

Copper concentration in livers of sheep fed added Cu was not

affected by added dietary Cu, but was higher than that of

controls and tended to increase with time. In Experiment 5

sheep depleted of Cu for 3 weeks were supplemented with up

to 80 mg/kg Cu from RG acetate for 10 or 20 days. Liver was

the only tissue sampled in which Cu accumulated and the 10-

day assay was optimum. Experiment 6 was a bioavailability

assay. Sheep were depleted for 3 weeks, liver biopsied to

determine initial Cu concentration and then supplemented for

10 days with the same Cu sources as in Experiment 3. The

assay was incapable of differentiating among sources

evaluated; however, surgery trauma during biopsy may have

confounded the results. These studies indicate that tissue

Cu accumulation can be used as a bioassay criterion for

determining Cu bioavailability in chicks. Results of sheep

studies, however, were inconclusive.


xiv















CHAPTER I
INTRODUCTION


Copper (Cu), an essential element for animals and

plants, is a member of group lB of the periodic table of

elements and consists of two natural isotopes: 63Cu and
65Cu (69.1 and 30.9%, respectively; NRC, 1977). The element

forms more than 200 minerals but occurs primarily as copper

sulfide deposits in igneous rocks (Georgievskii, 1982). Its

concentration in the earth's crust has been estimated at 50

mg/kg, with soil content ranging from 1 to 50 mg/kg with an

average of approximately 20 mg/kg (NRC, 1977).

The presence of Cu in living matter was first demon-

strated in the early 1800s when it was shown to be present

in both plant and animal tissues (Miller et al., 1979).

Copper concentrations in plant material have since been

shown to range from as low as 1 mg/kg to as much as 50 mg/kg

on a dry tissue basis (NRC, 1977). Animal tissues also show

a wide range of Cu concentrations; however, Cu concentra-

tions in the whole body of various species appear to be

relatively uniform, averaging 2 mg/kg on a fat-free basis.

Evidence for an essential physiological role for Cu in

animals was first provided by Hart et al. (1928) when they

demonstrated that both Cu and Fe were required for hemo-

globin formation in anemic rats. Three years later Cu








deficiency in grazing cattle was demonstrated in Florida

(Neal et al., 1931) and by 1939 "neonatal ataxia" (swayback)

of lambs (Bennetts and Chapman, 1937) and "falling disease"

of cattle (Bennetts and Hall, 1939) were both recognized as

Cu deficiency syndromes that could be prevented by Cu

supplementation. Since then Cu has been shown to be essen-

tial for normal growth, iron metabolism, skeletal develop-

ment, cardiovascular integrity, reproduction, pigmentation

and keratinization, and development and function of the

central nervous system.

The chance of a Cu deficiency occurring with resultant

economic losses has resulted in most livestock diets being

routinely supplemented with Cu. Because the effectiveness

of such supplements depends on the utilization of the

elements by the animal, careful consideration of the bio-

logical availability of supplements is required. However,

attempts to determine Cu bioavailability from inorganic

sources, using traditional methods, have met with limited

success (Miller, 1983). Bioassays are complicated by Cu

absorption and retention values that are low relative to

that of other elements, by interactions between Cu and other

dietary constituents, and by the fact that Cu does not have

a radioactive isotope with a sufficiently long half-life to

permit many of the metabolic studies required.

Recently, Black et al. (1984a, b) working with

manganese (Mn) demonstrated that tissue uptake of Mn from

high level, short term supplementation of dietary Mn was a









useful measure of relative bioavailability of inorganic

sources. Reports of Cu accumulation in the tissues of

animals fed high dietary levels of Cu (Dick, 1954; Hill and

Williams, 1965; Norvell et al., 1975; Jensen and Maurice,

1979) suggest that this technique may be useful in the study

of Cu bioavailability.

The experiments reported herein were conducted to

determine if tissue Cu accumulation in sheep and chicks fed

high dietary levels of Cu could be used as bioassay crite-

rion for determining relative Cu bioavailability from

inorganic sources.















CHAPTER II
REVIEW OF LITERATURE


Copper is a member of group IB of the periodic table of

elements. These elements, differing essentially only in the

number of electrons in the 3d shell, are known as transition

elements. In addition to the metallic form, Cu can exist in

the +1, +2, or +3 valence states, but more commonly occupies

the +2 state (Miller, 1979). In natural mineral states, Cu

occurs primarily as copper sulfide deposits in igneous

rocks. In biological systems, the cuprous (Cu+) and cupric

(Cu+2) forms predominate (Miller et al., 1979). The rela-

tive ease with which one form is converted to the other by

addition or release of an electron gives Cu great versa-

tility as an electron acceptor or donor (Frieden, 1968).

The presence of Cu in living matter was first demon-

strated in the early 1800s when it was shown to be present

in both plant and animal tissues (Miller et al., 1979).

However, it was not until a century later that Cu was shown

to be essential for animals (Hart et al., 1928). Copper

has been shown to be an essential component of numerous

oxidative enzyme systems including ascorbic acid oxidase,

tyrosinase, uricase, cytochrome oxidase, monoamine oxidase

and lysyl oxidase (Underwood, 1977). Copper concentrations

in the whole body of various animal species average 2 mg/kg









on a fat-free basis, with newborn and young animals having

higher concentrations than adults of the same species. The

distribution of total body Cu among tissues varies with

species, age and Cu status of the animal (Underwood, 1977).


Copper Metabolism


Absorption


Copper is absorbed from the stomach and small intestine

of most animals; however, the site of maximal absorption

varies slightly among species. In rats, Cu is absorbed in

the stomach (Van Campen and Mitchell, 1965) and small intes-

tine (Tompsett, 1940; Owen, 1964; Van Campen and Mitchell,

1965), with absorption being greatest in the stomach (Van

Campen and Mitchell, 1965). The small intestine is the

primary site of absorption in humans (Dunlap et al., 1974),

pigs (Bowland et al., 1961), hampsters (Crampton et al.,

1965) and chicks (Starcher, 1969). Considerable net absorp-

tion of Cu also occurs in the large intestine of sheep

(Underwood, 1977).

The mechanism of Cu absorption is not precisely known.

Present evidence indicates that intestinal absorption occurs

in two steps. One step involves the mucosal uptake of Cu

from the lumen, probably as a result of binding to sites on

cell surfaces or within the cells. The second step involves

transport to the serosal side and appears to be energy

dependent (Crampton et al., 1965). These two steps appear









to be independent since anoxia and 2,4-dinitrophenol inhibit

transport of Cu into serosal fluid, but have no effect on

mucosal uptake.

The extent of Cu absorption is influenced by the amount

and form of Cu ingested, by the dietary level of several

other metal ions and organic substances, and by the age of

the animal (Underwood, 1977). In a recent review, Cousins

(1985) examined effects of various dietary constituents on

Cu absorption and found that absorption is increased by

amino acids, nitriloacetate, citrate, phosphate, gluconate,

oxalate and ethylenediaminetetraacetic acid, while phytate,

ascorbic acid, thiomolybdate, fiber, bile and zinc decrease

absorption. The influence of dietary protein level on Cu

absorption is not as clearly defined. In some studies, high

protein diets have been shown to enhance Cu absorption

(Engel et al., 1967; Greger and Snedeker, 1980), while in

others, it appears to decrease absorption (McCall and Davis,

1961; Combs et al., 1966). Differences in protein digesti-

bility may account for some of these differences, since

digestibility could have a marked effect on the extent to

which copper-amino acid and peptide chelates are formed

(Cousins, 1985). Absorption of dietary Cu has been esti-

mated to be between 40 and 60% in humans (Mason, 1979.). In

contrast, Cu absorption is estimated to be less than 10% in

mature sheep (Suttle, 1972).









Transport


Plasma Cu is divided between two pools, one tightly and

one loosely bound. The plasma cuproprotein ceruloplasmin

represents the tightly bound pool that was thought to

account for at least 90% of total plasma Cu in most species

(Gubler, 1953). However, a recent report indicates that

ceruloplasmin may account for only 60 to 65% of plasma Cu in

rats and humans (Linder et al., 1985). The loosely bound

pool is made up of albumin and amino acid complexes of Cu

which are in equilibrium with each other and with ionic Cu

(Neumann and Sass-Kortsak, 1967). There appears to be very

little exchange between these two pools (Sternlieb et al.,

1961). Once absorbed, Cu is complexed with albumin and

amino acids and is transported via the hepatic portal vein

primarily to the liver (Harris and Sass-Kortsak, 1967;

Neumann and Sass-Kortsak, 1967). Recently, Linder et al.

(1985) reported an additional Cu transport protein in rat

and human serum. The protein, tentatively named

"Transcuprein," has a greater affinity than albumin for Cu.

However, in both in vitro and in vivo systems, it readily

exchanges Cu with albumin. In addition, it was shown to

donate its Cu to cultured cells more effectively than

albumin. The presence of this protein in serum has yet to

be confirmed by other researchers. Evans (1973) suggested

that, although amino acid bound Cu represents only a small

fraction of total plasma Cu, it may be a vital component of









the Cu transport mechanism since, in addition to facilitat-

ing Cu transport in blood, it also facilitates transport of

Cu across a number of cell membranes. Upon arrival at the

liver, a portion of complexed Cu is allowed to pass directly

to systemic circulation, where it constitutes about 7% of

plasma Cu. The remainder is taken up by hepatocytes and

bound to metallothionein in the cytosol (Mason, 1979; Weiner

and Cousins, 1980).

In liver, Cu can be stored, excreted in bile, or

incorporated into newly synthesized ceruloplasmin which is

discharged into plasma (Marceau and Aspin, 1972). The role

of ceruloplasmin in Cu transport has only recently been

demonstrated. In 1965, Owen demonstrated that Cu uptake by

tissues is closely related to release of ceruloplasmin from

liver, suggesting that ceruloplasmin transfers its Cu to

those tissues. Further evidence for a transport role was

furnished by Marceau and Aspin (1972) who showed that in

rats, ceruloplasmin and albumin transfer their Cu to

tissues of the body. Subsequently, Hsieh and Frieden (1975)

found that ceruloplasmin Cu more quickly restored tissue

cytochrome oxidase activity in deficient rats than did an

equal amount of a Cu-albumin solution. More recently,

Harris and DiSilvestro (1981) showed that activity of lysyl

oxidase in chick aorta is directly correlated with cerulo-

plasmin content of plasma. Campbell et al. (1981) found

that, in the rat, ceruloplamsin is the preferred plasma

source of Cu for normal and malignant cells, and that









ceruloplasmin Cu turns over more rapidly than the protein

moiety, a finding consistent with its role as a transport

protein.

In summary, it appears that absorbed Cu is transported

via the hepatic portal vein to liver complexed with albumin,

amino acids and perhaps transcuprein. In liver, cerulo-

plasmin is synthesized and released into peripheral plasma.

Plasma ceruloplasmin, albumin, amino acids and perhaps

transcuprein then transport Cu to tissues of the body.


Excretion


In view of the relatively poor absorption of dietary

Cu, it is not surprising that a high proportion of ingested

Cu appears in feces. Although much of the fecal Cu repre-

sents unabsorbed Cu, excretion of Cu occurs chiefly via the

biliary system (Gitlin et al., 1960; Beck, 1961; Bowland

et al., 1961; Cartwright and Wintrobe, 1964a, b). In addi-

tion to being directly involved in Cu excretion, bile also

appears to play an indirect role. Gollan et al. (1971)

demonstrated the ability of bile to bind Cu and suggested

that bile may also enhance Cu excretion by decreasing

absorption. The ability to inhibit Cu absorption was

demonstrated subsequently in rats (Gollan, 1975).

Under normal conditions, urinary excretion of Cu is

minimal. In humans it is estimated to be approximately .5

to 3% of daily Cu intake (Cartwright and Wintrobe, 1964b).

Evans (1973) attributed this to the fact that in blood the









majority of Cu is bound to ceruloplasmin or confined within

erythrocytes and cannot permeate the glomerular capillaries.

Urinary Cu excretion is increased in a number of human

diseases, including renal diseases, proteinuria, cystinuria,

diabetes and Wilson's disease (Owen, 1982b). In addition to

fecal and urinary loss, negligible amounts of Cu are lost

via sweat and normal menstrual flow.


Copper Deficiency and Functions


The manifestation of Cu deficiency varies with age, sex

and species of animal, and with severity and duration of the

deficiency (Underwood, 1977). Anemia is a common expression

of Cu deficiency in all species when deficiency is severe or

prolonged. Other Cu deficiency signs include skeletal and

central nervous sytem abnormalities, depigmentation of hair

and wool, infertility, cardiovascular diseases, diarrhea and

immunodeficiency (Miller et al., 1979). In ruminants,

physiological Cu deficiencies can be produced by four feed-

ing situations: (1) high molybdenum (Mo), generally above

100 mg/kg diet, (2) low Cu:Mo ratio, 2:1 or less, (3) Cu

deficiency, below 5 mg/kg diet and (4) high protein, 20 to

30% in fresh forage (Ward, 1978). This makes diagnosis and

treatment of a Cu deficiency in ruminants difficult.

The physiological function of a micronutrient, such

as Cu, can frequently be elucidated by studying the

pathology that results from a deficiency of the nutrient

(O'Dell, 1976). Most Cu deficiency diseases listed









previously can be related directly to a deficiency of

specific Cu-dependent enzyme systems. These systems and

their related diseases are discussed in the following

sections.


Anemia and Iron Metabolism


The first evidence that Cu plays an essential role in

animal physiology was provided by Hart et al. (1928) who

showed that it is required for prevention of anemia. Since

then, anemia has been found to be a common expression of Cu

deficiency in all species. In rats, rabbits, pigs and lambs

this anemia is hypochromic and microcytic, while in ewes and

cattle it is hypochromic and macrocytic (Underwood, 1977).

Anemias that are hypochromic and microcytic result from

defects in hemoglobin synthesis. The causes of defective

hemoglobin synthesis can be subdivided into three groups:

(1) those affecting iron (Fe) metabolism, (2) those affect-

ing biosynthesis of porphyrins and heme and (3) those

affecting biosynthesis of globin (Lee et al., 1976). A

role for Cu in the biosynthesis of globin has not been

established to date.

Morphologic and biochemical similarities between Cu

deficiency and anemia due to Fe deficiency led Lahey et al.

(1952) to suggest that in Cu-deficient swine there is an

abnormality in Fe metabolism. Evidence of abnormal Fe

metabolism was demonstrated subsequently by Gubler et al.

(1952). These researchers found that Cu-deficient pigs have









an impaired ability to absorb Fe, mobilize it from tissues

and utilize it in hemoglobin synthesis. Further evidence

for an impairment of mobilization from tissues was provided

by Lee et al. (1968b) when they demonstrated failure of the

duodenal mucosa, the reticuloendothelial system and hepatic

parenchymal cells to release Fe to plasma at the same rate

as control animals.

An important step toward an understanding of the role

of Cu in Fe metabolism was made by Osaki et al. (1966) when

they proposed that ceruloplasmin, by virtue of its ferroxi-

dase activity, could enhance the rate of Fe saturation of

transferrin and thus allow delivery of adequate amounts of

Fe to erythropoietic sites. This proposal appeared to be

validated when impairment in Fe metabolism was corrected

by intravenous administration of ceruloplasmin to Cu-

deficient pigs (Lee et al., 1968b; Roeser et al., 1970).

Administration of ceruloplasmin to Cu-deficient pigs with

normal Fe stores caused an increase in plasma Fe which was

proportional to the log of ceruloplasmin dose. However,

recent studies demonstrate, at least in rats, that it is

possible to have a reduction in ceruloplasmin levels and

still not develop anemia (Cohen et al., 1983; Fields et al.,

1984).

In addition to its role in mobilization of Fe for

hemoglobin formation, Cu appears to be essential for

normal normoblast function. Lee et al. (1968a) found that

Fe could not be incorporated into hemoglobin and, instead,









accumulated as nonhemoglobin Fe. Investigation of the heme

biosynthetic pathway shows no reduction in activity of any

heme biosynthetic enzymes. In fact, as anemia developed,

activity of heme biosynthetic enzymes increased, appearing

to indicate that Cu could not be a cofactor in these enzymes

(Lee et al., 1968a). A possible explanation of the normo-

blast defect was proposed by Goodman and Dallman (1969)

when they suggested that a Cu protein is required for

normal mitochondrial Fe uptake, a step essential for heme

synthesis. This proposal led a number of researchers to

investigate the role of mitochondria in heme synthesis.

Their observations indicate that an active, intact

mitochondrial electron transport system is necessary for

optimal synthesis of heme (Jones and Jones, 1969; Lemberg,

1969; Williams et al., 1976). Using Cu-deficient mito-

chondria with depleted cytochrome oxidase activity, Lee

et al. (1976) demonstrated that these mitochondria consumed

less oxygen and synthesized less heme than did normal

mitochondria. Further, the rate of heme synthesis corre-

lated with cytochrome oxidase activity. They therefore

proposed that the defect in heme synthesis in Cu-deficient

mitochondria is due to a deficiency of cytochrome oxidase.

This proposal received support recently when Davies et al.

(1985) demonstrated a marked decline in liver mitochondrial

cytochrome oxidase activity in Cu-deficient rats.








Pigmentation and Keratinization


The Cu enzyme tyrosinase catalyzes oxidation of the

amino acid tyrosine and other phenolic compounds to produce

melanin, a pigment responsible for darkening of skin, hair

and wool in most animals (Frieden, 1968). Mammalian

tyrosinase contains between .22 to .25% Cu and has a

molecular weight of about 33,000 daltons. Changes in hair

and wool color are among the most characteristic manifesta-

tions of a Cu deficiency in sheep and cattle. In fact, lack

of pigmentation in sheep has been reported to be a more

sensitive index of Cu deficiency than anemia (Underwood,

1977). Decreased feather pigmentation has also been

observed in chicks and poults fed Cu-deficient diets (Hill

and Matrone, 1961; Savage et al., 1966). Tyrosinase has

also been shown to be involved in albinism and a type of

skin cancer called melanoma. In albinism, there is an

absence or inactivity of tyrosinase, while in melanoma there

is hyperactivity of tyrosinase (Frieden, 1968).

Impaired keratinization, a specific effect of Cu

deficiency, is observed in a number of species. In sheep,

it is characterized by failure of wool fibers to crimp.

This failure is attributed to inadequate cross-linkage among

fibers and a reduced number of disulfide bonds (Underwood,

1977). Copper supplementation of deficient sheep results in

complete restoration of crimping.









Skeletal Abnormalities


Skeletal abnormalities resulting from Cu deficiency

have been reported in most species. The abnormalities vary

depending on species and on whether the deficiency is

primary or secondary in nature. In dogs (Baxter and Van

Wyk, 1953) and pigs (Follis et al., 1955), histological

changes include thinned cortices, broadened epiphyseal

cartilage and reduced osteoblastic activity. In chicks

(Carlton and Henderson, 1964), the widened epiphyseal

cartilages and thinner cortical bones observed in pigs and

dogs are present, but the widened calcified cartilage matrix

of the upper metaphysis is absent. Suttle et al. (1972)

found that lambs born to, and suckled by, Cu-deficient ewes

had none of the gross abnormalities observed in the dog, pig

or chick. There was no evidence of widened epiphyseal

cartilage observed in other species. The lesions observed

were attributed to reduced osteoblastic activity. Bone

abnormalities in cattle appear to differ depending on the

type of Cu deficiency. In a primary deficiency, there

appears to be a general matrix osteoporosis with decreased

osteoblastic activity (Suttle and Angus, 1978), while in a

secondary deficiency there is an irregular widening of

epiphyseal cartilage in the presence of active osteoblasts

(Irwin et al., 1974).

The defect in Cu-deficient bone is attributed to a

failure of collagen cross-linking resulting from a decreased









activity of amine oxidases. Rucker et al. (1969) found that

amine oxidase activity was reduced 30 to 40% in Cu-deficient

chicks. In addition, bone collagen contained less aldehyde

and was more soluble. Solubility of collagen was previously

shown to be inversely related to the degree of cross-linking

(Harding, 1965). Decreased cross-linking and aldehyde

content of Cu-deficient collagen has also been observed by

Chou et al. (1969). These researchers proposed that the

catalytic role of Cu is mediated through an amine oxidase

which catalyzes formation of an intermediate aldehyde

compound which is involved in intramolecular cross-links.

Further evidence for a role of Cu in cross-linking was

provided by Rucker et al. (1975) when they demonstrated that

Cu-deficient bones, which fractured with less deformity and

torque than control bones, could be strengthened to that of

control bones by addition of artificial cross-links.

Discovery of the amine oxidase, lysyl oxidase and the

elucidation of its functions provided a greater understand-

ing of the role of Cu in maintaining normal collagen

cross-links. This Cu enzyme was shown to oxidatively

deaminate lysyl and hydroxylysyl radicals in collagen,

forming allysine and hydroxylysine (Narayanan, 1978; Rucker

and Murray, 1978; Siegel, 1979). Allysine and hydroxylysine

then condense with other peptide allysines, lysine or

hydroxylysine residues to form cross-links (Opsahl et al.,

1982). Failure to form adequate cross-links because of









reduced lysyl oxidase activity results in the skeletal

abnormalities observed in most species.


Cardiovascular System


Cardiovascular defects associated with Cu deficiency

have been reported in cattle (Bennetts et al., 1948; Leigh,

1975), swine (Gubler et al., 1957; Shields et al., 1962),

chicks (O'Dell et al., 1961; Carlton and Henderson, 1963),

turkeys (Savage et al., 1966; Simpson et al., 1971), rabbits

(Hunt and Carlton, 1965), rats (Abraham and Evans, 1972;

Kelly et al., 1974; Wallwork et al., 1985) and guinea pigs

(Everson et al., 1967). Cardiac hypertrophy, a common

pathology of Cu deficiency, has been observed in cattle

(Leigh, 1975), swine (Gubler et al., 1957; Shields et al.,

1962) and rats (Abraham and Evans, 1972; Kelly et al., 1974;

Wallwork et al., 1985). Associated with cardiac hypertrophy

is an alteration in cardiac mitochondrial morphology

accompanied by decreased cytochrome oxidase activity.

Cytochrome oxidase is the terminal link in the electron

transport chain (Green, 1956) and is found primarily in

mitochondrial membranes of most tissues (Gallagher and

Reeve, 1976; Chance and Leigh, 1977). Reduced tissue

cytochrome oxidase appears to be a consistent finding in

Cu-deficiency (Owen, 1982a). In cattle, a condition known

as falling disease is characterized by sudden deaths,

usually after mild exercise or excitement. The disease

believed due to acute heart failure results from








degeneration of the myocardium with replacement fibrosis

(Underwood, 1977).

Extensive hemorrhaging resulting from rupture of major

blood vessels is observed in chicks (O'Dell et al., 1961),

pigs (Shields et al., 1962) and guinea pigs (Everson et al.,

1967). In all cases cardiovascular tissues appear to lose

the ability to maintain normal elasticity. Starcher et al.

(1964) found that aortas of Cu-deficient chicks contained

only 50% as much elastin as did control chicks by day 17.

O'Dell et al. (1966) found that Cu-deficient aortas

contained less elastin and more soluble collagen than did

controls. Copper-deficient elastin contained 40% less

desmosine and isodesmosine and five times as much lysine as

did the control protein. In Cu-deficient pigs (Shields

et al., 1962), a derangement of elastic membranes results in

a 60% decrease in the tensile strength of thoracic aortas.

In rabbits (Hunt and Carlton, 1965), elastin defects consist

of loss of elastic fibers and calcification and fragmenta-

tion of internal elastic membranes in muscular and elastic

arteries.

Observations of reduced elastin synthesis and changes

in the nature of the deficient protein suggested a role for

Cu in elastin synthesis.. Elucidation of such a role began

with the observation that Cu-deficient elastin contained a

high concentration of lysine, but a low concentration of

desmosine and isodesmosine (Starcher et al., 1964; Miller

et al., 1965; O'Dell et al., 1966). Desmosine and its









isomer isodesmosine are tetracarboxylic tetra-amino acids

involved in elastin cross-linkage (Thomas et al., 1963).

Based on structure, Partridge et al. (1964) suggested that

both amino acids could arise from condensation of four

lysine residues pre-existing in straight chain elastin

precursors. In order for condensation to occur the epsilon

amino group of lysine would have to be oxidatively

deaminated to an aldehyde. Such a reaction is catalyzed

by amine oxidases. Kim and Hill (1966) examined this

hypothesis and found that Cu deficiency resulted in reduced

oxidase activity in aortas. Addition of amine oxidase to

Cu-deficient systems decreased lysine-to-desmosine ratios

to approximately that of Cu supplemented systems. The

cuproenzyme, lysyl oxidase, was subsequently shown to be

the amine oxidase that catalyzes the oxidative deamination

of lysine to give allysine, the aldehyde derivative. In

elastin, three allysines combine with one lysyl to form

desmosine (Owen, 1982b).

In summary, the role of Cu in cardiovascular integrity

involves two Cu-dependent enzymes, cytochrome oxidase and

lysyl oxidase. Cytochrome oxidase is required for cardiac

mitochondrial integrity and lysyl oxidase is required for

synthesis of normal elastin.


Central Nervous System


Nervous disorders resulting from Cu deficiency are

observed in neonates of several species including sheep









(Bennetts and Chapman, 1937; Innes and Shearer, 1943; Barlow

et al., 1960a, b), goats (Owen et al., 1965; O'Sullivan,

1977; Inglis et al., 1986), rats (Carlton and Kelly, 1969;

Dipaolo et al., 1974), pigs (Joyce, 1955; Wilkie, 1959;

McGavin et al., 1962) and guinea pigs (Everson et al., 1967,

1968). The disorder observed most frequently occurs in

lambs and goats and has been termed neonatal ataxia or

"swayback." Swayback is characterized by motor dysfunction

of the hind limbs and is associated with subnormal levels of

Cu in tissues of ewes and lambs (Underwood, 1981). Two

forms of swayback occur in lambs and goats, a congenital

form in which the animals are affected at birth and a

delayed form in which signs may not appear for several

weeks.

The pathology of swayback was initially characterized

as a diffuse symmetrical demyelination of the cerebrum, with

liquefaction and cavitation being a common end stage of the

lesion (Innes and Shearer, 1943). This characterization of

swayback was challenged by Behrens and Schulz (1959) and

Schulz and Behrens (1960) who suggested that the disorder

resulted from venous stasis, edema, perivascular softening

and necrosis. Barlow (1958) and Barlow et al. (1960b) found

that although lesions of cerebral white matter were almost

always pathogonomic for swayback, they were not an essential

part of the disease. They concluded that neuronal hyaline

necrosis and fiber degeneration in the brain stem and spinal

cord was the essential pathology of swayback.









Recently, Howell et al. (1981) investigated cases of

congenital and delayed swayback and proposed a possible

explanation for observed differences in the pathology of

the disease. They proposed that pathological lesions would

differ depending on when the Cu deficiency occurred. A Cu

deficiency in early or mid pregnancy results in cerebral

lesions, since maximum cerebral growth is occurring during

this period. In late pregnancy, Cu deficiency results in

lesions of the cerebellum and of myelinated structures in

the cerebrum and cord. Copper deficiency occurring after

parturition affects the spinal cord most severely, since the

spinal cord of the lamb more than doubles in size from

5 weeks of age to maturity. There appears to be some

support for such a proposal since swayback can be prevented

either by supplementing pregnant ewes (Allcroft et al.,

1959) or by supplementing deficient lambs of highly

susceptible populations (Lewis et al., 1981).

Innes and Shearer (1943), after discussing the possi-

bility of myelin aplasia being involved in the pathogenesis

of swayback, concluded that the disease was the result of

demyelination. This conclusion appeared to receive support

from Barlow (1963) who showed that myelin present in the

brain of newborn lambs disappeared with age while products

indicative of myelin degeneration increased. However, in

the same study, Barlow (1963) pointed out that failure of

the mechanism of myelin fabrication or myelin degeneration

might both equally well account for the pathological









lesions seen. Since then several researchers have presented

evidence in favor of myelin aplasia. Howell et al. (1964)

found less myelin in the central nervous system of swayback

lambs and, since no products of myelin degeneration were

detected, concluded that the lesion was the result of myelin

aplasia. Everson et al. (1968), using the guinea pig as a

model, concluded that Cu deficiency affects normal develop-

ment of myelin throughout the brain. There was no evidence

of myelin degeneration. Dipaolo et al. (1974) found histo-

logical evidence for decreased myelination and demonstrated

significant reductions in typical myelin lipids in

Cu-deficient rats. Zimmerman et al. (1976) found a 56%

decrease in yield of myelin per gram of brain tissue in

22-day-old rats whose dams were Cu-deficient. The absence

of cholestrol ester in myelin from Cu-deficient animals

suggested that there was no myelin degeneration.

Observations of abnormal myelination in Cu-deficient

animals implied a role for Cu in myelin synthesis.

Gallagher et al. (1956a, b) provided the first evidence for

such a role when they demonstrated loss of activity of the

cuproenzyme cytochrome oxidase and suppression of phospho-

lipid synthesis in Cu-deficient rats. As a direct result of

these observations, Howell and Davidson (1959) examined

brain tissue from swayback lambs and found subnormal levels

of Cu and cytochrome oxidase. Barlow (1963) confirmed these

findings and also found that enzyme activity was lowest in

those sites in which lesions of the disease appear.









Similar findings led Fell et al. (1965) to suggest that a

low Cu content in brain leads to a deficiency of cytochrome

oxidase. Gallagher and Reeve (1971) supplied the final

evidence when they postulated that loss of cytochrome

oxidase activity leads to depressed phospholipid synthesis

by liver mitochondria, by interfering with the provision of

endogenous ATP to maintain an optimal rate of synthesis.

This hypothesis received support recently when Davies et al.

(1985) found that ATP and ADP contents of Cu-deficient

mitochondria were reduced by 39 and 40%, respectively.

Depressed phospholipid synthesis results in decreased myelin

synthesis since myelin is composed largely of phospholipids.

In addition to its role in myelination, Cu is required

for biosynthesis of the catecholamine neurotransmitters,

dopamine and norepinephrine. Significant depression in

catecholamine concentrations has been observed in ataxic

lambs (Hunt and Johnson, 1972). Parkinson's disease, an

extapyramidal disorder involving motor dynfunction in humans

is associated with depressed dopamine levels (Hornykiewicz,

1973). These observations suggest that impaired

catecholamine synthesis may be involved in the locomotor

disorder of swayback.

The precise mechanism by which Cu deficiency depresses

catecholamine synthesis is unknown. Tyrosine hydroxylase,

the rate limiting enzyme in dopamine synthesis, is signifi-

cantly depressed in Cu deficiency (Morgan and O'Dell, 1977).

The enzyme does not appear to be Cu-dependent (Nagatsu









et al., 1964) and its activity is not restored by Cu

repletion (Feller and O'Dell, 1980). This led Feller and

O'Dell (1980) to suggest that impaired dopamine metabolism

is due to a neuron development defect and not to a catalytic

dysfunction.

Norepinephrine is synthesized from dopamine in a

reaction catalyzed by the cuproenzyme dopamine-b-

hydroxylase (DBH). Depressed DBH activity could account

for the low nonepinephrine levels observed in Cu deficiency.

However, current evidence appears to indicate that this is

not the case. Dopamine-b-hydroxylase activity was found

to be significantly elevated in brains of rats made Cu-

deficient either by dietary means or as a result of a

genetic defect (Hunt, 1977; Prohaska and Smith, 1982).

Hesketh (1980, 1981a, b) investigated the effect of Cu

deficiency on adrenal glands of cattle and rats. In both

species there is a reduction in norepinephrine levels.

Dopamine-b-hydroxylase activity, however, is elevated in

rats but is unchanged in cattle. Hesketh (1981b) concluded

that depressed norepinephrine levels cannot be accounted for

only by reduced DBH activity but that it may be due to

defective storage, increased catabolism or reduced synthesis

of the catecholamine. Prohaska and Smith (1982) provided a

possible explanation for increased DBH activity. They

suggested that Cu deficiency, both dietary and genetic,

leads to reduced DBH activity in vivo, resulting in

reduced norepinephrine synthesis. However, depletion of









norepinephrine induces more apo-DBH synthesis, which is

then converted to DBH in vitro by trace amounts of Cu used

in the DBH assay. This explanation appears plausible since

Hesketh et al. (1985), using radioisotopes to study in vivo

catecholamine synthesis, concluded that DBH activity is

reduced in the norepinephrine-synthesizing cells of the

adrenal medulla of Cu-deficient rats. At present, there

is insufficient evidence to determine whether lower

catecholamine levels are causally related to swayback.


Reproduction


Impaired reproduction resulting from Cu deficiency was

first reported in rats by Keil and Nelson (1931). Rats fed

a milk diet, or a milk diet plus Fe produced only one litter

in 5 months. In comparison, rats fed the same milk and Fe

diet supplemented with .5 mg of copper sulfate daily pro-

duced an average of five litters. Dutt and Mills (1960)

also reported reproductive failure in Cu-deficient rats.

Out of 18 rats fed a low Cu diet, only one produced live

young. The rats cycled and conceived normally but then

resorbed their fetuses. In addition to fetal resorption,

Hall and Howell (1969) reported depigmentation of hair and

incisor teeth and a 90% reduction in liver Cu concentrations

of deficient rats.

Investigation of fetal resorption indicates that

fetuses and placentas of Cu-deficient rats develop normally

until day 13 of pregnancy. On day 13, fetal necrosis occurs









and is followed by placental necrosis on day 15. No lesions

were observed in the maternal ovaries, mammary or pituitary

glands (Howell and Hall, 1969). Fetal resorption appears to

occur only in severe cases of Cu deficiency. O'Dell et al.

(1961) were able to obtain litters from rats reared on a low

Cu diet containing about 1 mg/kg Cu, but the offspring were

severely anemic and nonviable. Lesions observed in the

newborn included severe edema, wide-spread subcutaneous

hemorrhage, skeletal abnormalities and abdominal hernias.

Copper deficiency in poultry results in egg abnormali-

ties and reduced hatchability. Laying hens fed a Cu-

deficient diet (.7-.9 mg/kg) for 20 weeks produced fewer

eggs and had subnormal concentrations of Cu in plasma, liver

and eggs. Hatchability of eggs dropped rapidly and by week

14 approached zero (O'Dell, 1968). Reduced hatchability was

attributed to early embryonic mortality. Copper-deficient

embryos were anemic and had a high incidence of hemorrhage

after 72-96 hours of incubation. Embryonic mortality was

also observed by Simpson et al. (1967). Feeding a Cu-

deficient diet (2 mg/kg) to laying hens resulted in

mortality of all embryos within 10 days. Microscopic

examination revealed that cells in the tunica media of

Cu-deficient embryos were immature and elastin synthesized

was abnormal. Baumgartner et al. (1978) fed hens a diet

containing .72 mg/kg Cu and observed that of eggs produced,

10% were shell-less and 40% were malformed. There was a

significant reduction in shell weight and thickness and









amino acid analysis of shell membranes indicated increased

lysine content.

Reproductive anomalies have also been reported in Cu

deficient ruminants. One of the first reports to implicate

Cu deficiency in reproductive disorders of ruminants was

that of Bennetts and Chapman (1937). They reported that

neonatal ataxia, a disease affecting lambs during gestation,

was caused by Cu deficiency and could be prevented by Cu

supplementation of pregnant ewes. Since then there have

been a number of reports associating Cu deficiency with

reproductive disorders. Howell (1968) reported that of nine

ewes fed a synthetic Cu-deficient diet, five died without

becoming pregnant, two aborted small, dead, immature fetuses

and two failed to conceive by the end of the experiment.

Anke (1973) found that Cu-deficient goats not only had lower

conception rates, but of those conceiving, 50% aborted.

Aborted fetuses were mummified and there was placental

necrosis. Wiener and Sales (1976) reported that Cu

deficiency did not appear to affect libido of rams, but rams

having higher plasma Cu concentrations appeared to be more

fertile.

The role of Cu in bovine reproduction is controversial.

Early reports appeared to indicate a negative relationship

between Cu deficiency and fertility (Allcroft and Parker,

1949; Blakemore and Venn, 1950; Becker et al., 1953;

Munroe, 1957). Low fertility was associated with reduced

conception rates resulting from delayed or depressed estrus.









Recent reports, however, implicate high molybdenum concen-

trations rather than Cu deficiency (Case et al., 1973;

Peterson and Waldren, 1977; Phillippo et al., 1982, 1985).

Phillippo et al. (1985) showed that a diet containing 5 ppm

molybdenum can delay the onset of first estrus in cattle

by 6-8 weeks. Associated with delayed estrus was a 50%

decrease in conceptions. These observations suggest that

earlier reports of impaired fertility in cattle may well be

molybdenum-induced, since the studies were conducted in

soils known to be high in molybdenum.

Studies relating blood Cu status to fertility are also

inconclusive. Some researchers found no relationship

between blood Cu and fertility (Rowlands et al., 1977;

Larson et al., 1980; Phillippo et al., 1982; Whitaker, 1982)

while others observed reduced fertility in animals exhibit-

ing low blood Cu concentrations (Bennetts et al., 1948;

Allcroft and Parker, 1949; Blakemore and Venn, 1950; Munroe,

1957). Similarly, Cu supplementation improved fertility in

some herds (Blakemore and Venn, 1950; Munroe, 1957;

Pickering, 1975; Hunter, 1977) but had no effect in others

(Poole and Walshe, 1970; Phillippo et al., 1982; Whitaker,

1982). These results indicate the need for definitive

studies on the role of Cu in bovine reproduction.

The precise lesions responsible for reproductive

abnormalities are yet to be elucidated. However, observa-

tions of fetal death associated with hemorrhaging and

placental necrosis suggest decreased vascular integrity of









blood vessels in the placenta and fetus. The role of Cu in

maintaining cardiovascular integrity has been discussed

previously.


Copper Toxicosis


Copper, although an essential element, can be toxic if

consumed in excess. Attempts to determine maximum tolerable

levels are complicated by numerous interrelationships among

Cu and other trace elements and substances. The situation

is further complicated by differences in susceptibility

between ruminants and nonruminants. In general, ruminants

are more susceptible to Cu toxicosis than nonruminants.

Like Cu deficiency, the basis for Cu toxicosis may well

represent diminished enzymatic activity since ionic Cu

inhibits a number of enzymes (Owen, 1981). The present

review will be limited to those species in which Cu toxi-

cosis has been reported to occur under practical feeding

conditions.

Reports of Cu toxicosis in swine first appeared in the

late 1950s. Not coincidentally, these reports began to

appear shortly after it was demonstrated that daily gains

of growing pigs were significantly improved by feeding 250

mg/kg Cu as copper sulfate (Barber et al., 1955a, b;

Bowler, 1955). Gordon and Luke (1957) diagnosed Cu toxi-

cosis as cause of death in a pig submitted for postmortem

examination. Necropsy revealed a generalized icterus and

all organs and tissues were stained an intense yellow.









Dietary Cu concentrations were unknown but liver analysis

revealed a Cu content of 2500 mg/kg on a dry matter basis

(DM). O'Hara et al. (1960) fed pigs a diet containing 250

mg/kg Cu as copper sulfate and reported incidences of

parakeratosis and a syndrome thought to be chronic Cu

toxicosis. Lesions were typical of chronic Cu toxicosis

reported in other species except for the absence of icterus

and hemoglobineria. Liver Cu concentrations of affected

pigs varied between 684 and 1800 mg/kg (DM). Copper levels

of 250 mg/kg and above also proved toxic in weanling pigs

fed a typical corn-soybean meal diet (Wallace et al., 1960).

However, in pigs fed a barley, wheat and fish meal diet, Cu

toxicosis did not occur until dietary levels exceeded 500

mg/kg Cu (Allcroft et al., 1961). Buntain (1961) found that

dietary Cu levels as low as 130 mg/kg may cause toxicosis

under certain conditions. During a 5 to 6 week period, 23

fattening pigs were lost from a herd of 200. Postmortem

examination revealed evidence of icterus, liver necrosis and

kidney damage. Liver Cu concentrations ranged from 1500 to

2200 mg/kg (DM).

Failure to establish an absolute toxic level for Cu in

swine suggests that factors other than dietary Cu level

may also influence the onset of toxicosis. Dietary levels

of zinc (Zn) and Fe are two such factors. Suttle and Mills

(1966a, b) reported that addition of 150 mg/kg Zn and 150

mg/kg Fe to diets containing 250 or 425 mg/kg Cu prevented

Cu toxicosis that developed when Zn and Fe were not added.









In swine fed 750 mg/kg Cu addition of 750 mg/kg Fe com-

pletely eliminated signs of toxicosis, while 500 mg/kg Zn

eliminated all signs except that of anemia. DeGoey et al.

(1971) found that Zn and Fe at dietary levels of 100 mg/kg

prevented signs of toxicosis in swine fed 500 mg/kg Cu.

The interaction of Cu, Zn and Fe in mammalian species has

been reviewed extensively by Davis (1980). He concluded

that diets that are either deficient in or contain excessive

levels of Cu, Zn or Fe will cause changes in the absorption

of the elements from the gastrointestinal tract. In swine,

Zn and Fe appear to alleviate Cu toxicosis by causing a

reduction in Cu absorption.

Suttle and Mills (1966a) investigated the etiology of

Cu toxicosis in swine. Liver damage was a consistent find-

ing in affected animals. The damage to liver tissue did not

appear to be caused directly by excessive storage of Cu,

since Cu concentrations in livers of affected animals were

similar to that of healthy Cu-supplemented animals. They

proposed that liver damage occurs when serum Cu levels

exceed 300 ug/100 ml. Below this level, liver is capable of

removing Cu from blood and storing it. Above this level,

liver tissue is probably damaged by excess Cu arriving at

the liver. This critical level was chosen since it was

found to be exceeded frequently in pigs with Cu toxicosis,

but rarely exceeded in healthy animals.

Copper toxicosis in poultry was investigated by several

groups. Pullar (1940) found the minimum lethal dose (MLD)









of Cu varied from 300-1500 on a mg/kg live body weight

basis, depending on class of poultry and form of Cu fed.

The maximum daily intake of copper carbonate tolerated by

birds was determined to be 60 mg/kg live body weight for

chicks, and 29 mg/kg live body weight for ducks. Overall,

ducks proved to be more sensitive to Cu toxicosis than were

pigeons or chickens. Goldberg et al. (1956) fed adult

leghorn hens increasing amounts of copper acetate (28-56

mg/kg live body weight) and observed weight loss, hemolytic

anemia and mortality within 5 weeks. Laying hens fed diets

containing more than 300 mg/kg Cu produced fewer eggs and

had lower liver lipid concentrations compared to controls

(Jackson and Stevenson, 1981a, b). Mayo et al. (1956)

reported reduced performance and muscular dystrophy in

chicks fed a corn-soybean meal diet supplemented with 324

mg/kg Cu. Smith (1969) observed a 7.5% reduction in live

weight gain of chicks fed 350 mg/kg Cu. In contrast,

Mehring et al. (1960) found 500 mg/kg Cu to be the minimum

toxic level in chicks fed a corn-soybean meal diet, while

Arthur et al. (1958) found no signs of toxicosis in chicks

fed 500 mg/kg Cu. Turkey poults appear to be more tolerant

of Cu than chicks. Wiederanders (1968) injected three

groups of poults with .5 mg Cu/day for up to 84 days and was

unable to produce Cu toxicosis until the dose was increased

to 5 mg/day for a further 17 days. Vohra and Kratzer (1968)

reported no deleterious effects in turkey poults fed 676

mg/kg Cu for 21 days. Christmas and Harms (1979) observed









reduced performance in poults fed 500 mg/kg Cu for 21 days,

but attributed this to the fact that the basal diet was

deficient in sulfur amino acids.

Like swine, dietary factors other than absolute Cu

levels may dictate the onset of Cu toxicosis in poultry.

Sulfur amino acid content of the diet appears to be of

prime importance. Several researchers (Jensen and Maurice,

1979; Christmas and Harms, 1979, 1984; Ekperigin and Vohra,

1981a) have demonstrated the ability of methionine to

alleviate the growth depressing effect of Cu. Robbins and

Baker (1980a, b) reported that feeding chicks 250 and 500

mg/kg Cu increased their sulfur amino acid requirement by

13 and 30%, respectively. Thus, minimum toxic levels of Cu

may have to be defined in terms of sulfur amino acid content

of diets.

Of all species, sheep are the most susceptible to Cu

toxicosis and can be affected at any age although most

reported cases occur in mature animals (Todd, 1969). Young

calves appear to be as susceptible as sheep, but become

more tolerant as they mature. With the exception of experi-

mentally induced Cu toxicosis (Kidder, 1949; Shand and

Lewis, 1957; Weiss and Baur, 1968) and a few cases of acute

toxicosis (Garner, 1961), there are very few reports of

chronic Cu toxicosis occurring in cattle under natural

grazing conditions. This led Todd (1969) to conclude that

from a nutritional point of view, chronic Cu toxicosis can

be considered as being almost entirely confined to sheep.









Causes of Cu toxicosis in sheep are myriad and include

oversupplementation of Cu, accidental use of high-copper

swine feed, use of Cu-based fungicides, overtreatment of Cu

deficiency and grazing of pastures deficient in molybdenum

or high in heliotropic species (Owen, 1981).

Copper toxicosis can be acute or chronic. Signs of

acute toxicosis include nausea, salivation, violent

abdominal pain, convulsions, paralysis, collapse and death.

Lesions observed on postmortem examination include gastro-

enteritis, necrotic hepatitis, splenic and renal congestion

and evidence of antemortem intravascular coagulation (NRC,

1980). In cases of acute toxicosis, death can occur within

24 to 72 hours (Isael et al., 1969; Wiener and MacLeod,

1970) or may be delayed 3 to 7 days (Macleod and Watt, 1970;

Sasu et al., 1970) depending on Cu dose and mode of

supplementation.

In contrast, cases of chronic toxicosis may not result

in mortality for several months. Chronic Cu toxicosis can

be divided into three distinctive phases, pre-hemolytic,

hemolytic and post-hemolytic. In the pre-hemolytic phase,

which can last from weeks to several months, signs of the

disease are absent, but Cu accumulates predominantly in

liver, and isolated hepatic parenchymal cells die liberating

their enzymes into the bloodstream (Howell and Kumaratilake,

1985). Hepatic Cu concentrations as high as 4000 mg/kg on a

dry matter basis have been reported (Gooneratne et al.,

1979). During the latter part of the phase there are marked









increases in serum concentrations of several enzymes includ-

ing glutamic oxaloacetic transaminase, lactic dehydrogenase,

glutamic dehydrogenase, sorbitol dehydrogenase and arginase

(Owen, 1981). Serum concentrations of Cu remain normal

until the hemolytic crisis is imminent, then sharply

increase (Eden, 1940; Todd and Thompson, 1963; Ishmael

et al., 1972). The transition from the pre-hemolytic to

the hemolytic phase is abrupt. Animals undergoing a

hemolytic crisis become dull, refuse to eat and are

excessively thirsty. There is a marked increase in blood

Cu which causes hemolysis, jaundice, methemoglobinemia,

hemoglobinuria and extensive necrosis of liver and kidney

tissue (Miller, 1979). In addition, there is evidence of

brain (Doherty et al., 1969; Howell et al., 1974) and muscle

(Thompson and Todd, 1974; Gooneratne and Howell, 1980)

damage. During the post-hemolytic phase, if the animal

survives, reparative changes commence but further episodes

of hemolysis can occur even if the source of Cu is removed

(Ishmael et al., 1971).

Recently, Gooneratne and Howell (1985) investigated the

cellular pathology of hepatic and renal cells in chronic Cu

poisoning of sheep and proposed the following pathogenesis

During Cu loading most of the excess Cu is stored
in liver lysosomes. In the early stages, liver
accommodates for increased hepatic Cu by prolif-
eration of its lysosomes. Beyond a 'critical'
hepatic Cu concentration, the rate of prolifera-
tion of lysosomes decreases and excess Cu is found
in enlarged, irregular lysosomes. At a further
'critical' Cu concentration the liver lysosomes
can no longer handle the increased hepatic Cu









uptake and they may rupture releasing Cu and acid
hydrolases into the cytoplasm, with resultant
liver cell death. Some of the Cu from the
released liver lysosomes enters the bloodstream
where it is transported in the plasma. Some of
this Cu enters the cells of the proximal con-
voluted tubules. The entry of Cu and acid
hydrolases into the already Cu loaded cells of
the proximal convoluted tubules may overload
their lysosomal system and be directly respon-
sible for the death of these cells. (1985,
p. 189)

Diagnosis of Cu toxicosis is a prerequisite for treat-

ment of the condition. Ross (1966) demonstrated the value

of the glutamic oxaloacetic transaminase assay as an aid in

the diagnosis and assessment of treatment of Cu toxicosis in

lambs. Once diagnosed, appropriate remedial steps can be

taken. The interaction among copper, molybdenum and sulfate

in ruminants has provided a basis for treatment of Cu

toxicosis. Dick (1956) reported that dietary sulfate

potentiated a copper-molybdenum antagonism which depressed

tissue Cu concentrations in ruminants. The precise

mechanism of interaction among these elements is still being

investigated but the hypothesis proposed by Suttle (1974b)

seems plausible. Dietary molybdate is thought to react with

sulfide (provided by reduction of sulfate) in the rumen to

form thiomolybdate. Thiomolybdate thus formed can then

interact with Cu either in the rumen or in tissues to form

complexes in which Cu is unavailable. Formation of such

complexes should undoubtedly prove helpful in alleviating Cu

toxicosis. Reports indicate that this may indeed be the

case. Suttle (1979) fed thiomolybdate to lambs and found









that hepatic accumulation of Cu was decreased by about 30%

in animals supplemented with Cu. Gooneratne et al. (1981a)

found that intravenous injections of 100 mg of ammonium

tetrathiomolybdate twice daily not only prevented hemolysis

but also prevented further crises in sheep already in

hemolysis. The authors concluded that chronic Cu toxicosis

can be successfully prevented or treated by intravenous

injections of appropriate doses of ammonium

tetrathiomolybdate.


Copper Bioavailability


Modern analytical techniques and instrumentation make

it possible for nutritionists to accurately determine

trace element concentration in animal tissues and feeds.

Unfortunately, however, such determinations provide no

information on utilization of the elements by animals.

Utilization involves the concept of bioavailability and is

critical in the selection of a suitable source of the

element. In terms of trace elements, bioavailability may

be defined as the proportion of an ingested element that is

absorbed, transported to its site of action, and converted

to the physiologically active species (O'Dell, 1983).

Traditionally, trace element bioavailability studies have

been conducted at deficient dietary levels, usually with

purified or semipurified diets. The mineral sources are

then added at graded levels and response criteria measured.

In general, the bioavailability of an element in a









particular source is determined relative to its functional

availability from a standard source. Use of a standard

source allows expression of bioavailability in terms of

relative biological availability (Miller, 1983). Since

cases of Cu toxicosis and deficiency are both often reported

in the literature, it is evident that the relative bioavail-

ability of Cu sources is of major concern.

Copper bioavailability studies were first conducted

using anemic rats. In these studies hemoglobin regeneration

was used as the response criterion. Schultze et al. (1934,

1936) found that Cu from copper caseinate, copper aspartate,

copper citrate, copper nucleinate, copper pyrophosphate,

cysteine cuprous mercaptide, wheat germ, brewers' yeast,

porcine liver and heart was readily utilized while that from

copper hematoporphyrin was not. In a comparison of Cu

salts, Cu from copper sulfate, cuprous oxide, cuprous iodide

and copper hydroxide was readily utilized by anemic rats.

Copper from cupric sulfide was found to be less efficiently

utilized (Keil and Nelson, 1931; Sherman et al., 1934).

In addition to hemoglobin regeneration, melanin-pigment

regeneration, growth and liver Cu storage in rats have also

been used as indexes of bioavailability. Mills (1955, 1956)

reported that Cu from aqueous extracts of herbage was more

available than Cu from copper sulfate. The author concluded

that the greater availability of Cu from herbage extracts

resulted from more efficient transport of herbage extract Cu

through the intestinal mucosa.









In studies with mature rats and using liver Cu storage

as the criterion, Nesbit and Elmslie (1960) found copper

oxide and copper pyrophosphate to be 27 and 49% as avail-

able, respectively, as was copper sulfate. Lo et al.,

(1984) evaluated availability of Cu from isolated soybean

protein and found it to be as available as Cu from a

standard copper carbonate source. Serum and liver Cu con-

centrations were both effective indexes of bioavailability.

Price and Chesters (1985) recently reported the development

of a new bioassay for assessment of Cu availability. The

technique involves a period of depletion followed by reple-

tion and is based on the observation that increases in

cytochrome oxidase activity in the duodenal mucosa of

partially Cu depleted rats is linearly related to Cu dose.

Using this technique, the authors found that Cu from

untreated dried grass, rumen, duodenal and ileal digest of

sheep were 75, 12, 43, and 28% as available, respectively,

as that of copper sulfate.

Compared to rats, there are fewer reports of Cu bio-

availability studies in poultry. Willingham and Hill (1970)

compared oxide and carbonate forms of Cu to that of the

sulfate form. They found that chicks fed the sulfate form

grew faster and had higher liver Cu and hemoglobin concen-

trations at day 21, than those fed oxides or carbonates.

McNaughton et al. (1974) found cuprous oxide and cuprous

iodide to be 76 and 82% as available, respectively, as

copper sulfate, in broilers fed test diets for 21 days.









Physiological indicators used to determine bioavailability

included hematocrit, hemoglobin and liver Cu concentrations.

Norvell et al. (1975) fed graded levels of Cu (120-720

mg/kg) as the acetate, chloride and sulfate salts to broiler

chicks for periods up to and including 8 weeks of age. They

found that Cu in the acetate, chloride and sulfate forms

depressed gains and increased Cu residues in liver, muscle

and kidney tissue. Comparable levels of Cu fed as the oxide

salt had no effect on these parameters, indicating that Cu

oxide was less available than the other salts.

As with poultry, few Cu bioavailability studies have

been conducted in swine. Copper as cupric sulfate is more

efficiently absorbed and stored in porcine liver than is Cu

as cupric sulfide (Barber et al., 1961; Bowland et al.,

1961; Cromwell et al., 1978). Buescher et al. (1961) fed

pigs radioactive Cu in the form of cupric carbonate, cupric

sulfate or cuprous oxide and based on serum uptake and

excretion of radiocopper concluded that the biological

availabilities of the three forms were similar for swine.

Copper carbonate was found to be equally as effective as

copper sulfate in promoting growth in pigs fed 250 mg/kg Cu,

but was less effective in promoting liver Cu storage (Allen

et al., 1961). In weanling pigs fed 250 mg/kg supplemental

Cu as copper sulfate, copper oxide or copper carbonate,

liver Cu concentrations were highest in those fed sulfate

(218 mg/kg), lowest in those fed oxide (32 mg/kg), and









intermediate in those fed carbonate (89 mg/kg; Bunch et al.,

1961, 1965).

Copper bioavailability studies in ruminants have been

conducted in both sheep and cattle. Lassiter and Bell

(1960) used sheep to determine the relative availability

of 64Cu from cupric sulfate, cupric chloride, cupric oxide

needles and powder, cuprous oxide, cupric nitrate, cupric

carbonate and copper wire. Copper from the powdered oxide

was more available than from the needles, while Cu from

wire was essentially unavailable. Cupric sulfate, cupric

chloride and cupric nitrate had similar availabilities.

Copper absorption was found to be higher from oral doses

of cupric carbonate than from cupric or cuprous oxide.

Suttle (1974a), noting that conventional balance techniques

are inadequate to determine Cu availability because only a

small proportion of Cu ingested by sheep is absorbed (10%

or less) and differences in absorption can be masked by

analytical errors (Mills and Williams, 1971), proposed the

use of a depletion-repletion technique. With this technique

depleted sheep are repleted with oral and intravenous doses

of Cu. The relative responses of plasma Cu to oral and

intravenous Cu is then used to estimate true availability of

dietary Cu. Using this technique Suttle (1974) estimated

the availability of dietary Cu to be 4.1% in one experiment,

while in a second experiment availabilities ranged from 4.5

to 11.4%. The author concluded that this technique provides

a sensitive method for assessing the availability of Cu in









ruminant diets. Chapman and Bell (1963) used a radioisotope

technique to evaluate the relative absorption and excretion

of Cu from various sources by beef cattle. Copper from

cupric nitrate, cupric sulfate and cupric chloride was

absorbed to a similar extent. Cupric carbonate had the

highest rate of absorption but also had the highest rate of

excretion, indicating that retention may be a problem.

Copper from powdered cupric oxide was absorbed to a greater

extent than Cu from cupric oxide needles. Absorption,

although not a true measure of bioavailability, is a major

component of bioavailability and as such does provide an

indication of bioavailability. The most recent report of Cu

bioavailability in ruminants was that of Kincaid et al.

(1986) who reported that Cu from copper proteinate was more

available than Cu from copper sulfate for calves fed diets

containing copper-molybdenum ratios varing from .9 to 2.8.

In addition to in vivo studies, several in vitro

techniques have been developed in an attempt to predict

bioavailability. Solubility tests have proved by far to be

the most effective. Solvents evaluated include distilled

water, hydrochloric acid, citric acid and neutral ammonium

citrate. Of these, the hydrochloric acid test has become

one of the most widely used procedures in the feed industry

(Bernhardt, 1986).

Information presented in this review demonstrates not

only the essentiality of Cu, but also the consequences of

deficiency and toxicity. It is also evident that although









relative bioavailability of Cu sources is of major concern,

relatively few Cu bioavailability studies have been

conducted. Mills and Williams (1971) may have provided an

explanation for this when they noted that conventional

balance techniques are inadequate to determine Cu avail-

ability as only a small proportion of ingested Cu is

absorbed and differences in absorption can be masked by

analytical errors.

In a 1983 symposium on the bioavailability of essential

and toxic trace elements, O'Dell (1983) cited the need for

the development of simplified assays for determining trace

element bioavailability. Recently, Black et al. (1984a, b),

working with manganese (Mn) demonstrated that tissue uptake

of Mn from high level, short term supplementation of dietary

Mn was a useful measure of relative bioavailability of

inorganic sources. Reports of Cu accumulation in the

tissues of animals fed high dietary levels of Cu (Dick,

1954; Hill and Williams, 1965; Norvell et al., 1975; Jensen

and Maurice, 1979) suggest that this technique may be useful

in the study of Cu bioavailability.















CHAPTER III
EFFECT OF HIGH DIETARY COPPER ON TISSUE
MINERAL COMPOSITION OF BROILER AS A
BIOASSAY OF CU SOURCES--EXPERIMEN1 1


Introduction


Attempts to determine Cu bioavailability from inorganic

sources, using traditional methods, have met with limited

success (Miller, 1983). Bioassays are complicated by the

facts that Cu absorption and retention values are low rela-

tive to that of other elements, and that interactions exist

between Cu and other dietary constituents. In addition,

Miller (1979) noted that Cu does not have an isotope with a

sufficiently long half-life to permit many of the metabolic

studies required.

Black et al. (1984a, b) working with manganese (Mn),

found that tissue Mn accumulation during short term high

level supplementation of the element can be used as a

bioassay criterion in determining Mn availability from

inorganic sources. This technique, described in detail by

Henry et al. (1987), has several advantages over traditional

techniques including the use of natural diets which are less

expensive and which allow the animal to express its maximum

genetic growth potential. With this technique if tissue

mineral accumulation (or its transformation) is linearly









related to and correlated with dietary concentration of the

element, then bioavailability can be determined by comparing

test sources to a standard source using slope-ratio methods.

Reports of Cu accumulation in the tissues of chicks fed

high dietary concentrations of Cu (Norvell et al., 1975;

Jensen and Maurice, 1979; Robbins and Baker, 1980a, b)

suggest that the technique of Black et al. (1984a, b) may be

of some utility in the study of Cu bioavailability.

This experiment was conducted to determine the rela-

tionship between dietary Cu and tissue Cu accumulation and

to evaluate the relative value of various tissues for use in

a bioavailability assay.


Materials and Methods


One hundred and sixty-eight, day-old Cobb feather-sexed

male chicks were assigned randomly to pens in a thermo-

statically controlled electrically heated Petersime battery

with raised wire floors. Chicks were maintained on a 24 h

constant-light schedule and allowed ad libitum access to

feed and tap water.

A completely randomized design was used with six

replicates of seven chicks assigned to each of four dietary

treatments. Dietary treatments were the basal diet (Table

3-1) supplemented with 0, 100, 200 or 300 mg/kg Cu as

reagent grade cupric acetate. The basal diet contained

10.5 mg/kg Cu (dry basis) by analysis and was formulated to

meet the requirements of growing chicks (NRC, 1984).









TABLE 3-1. COMPOSITION OF BASAL DIET--EXPERIMENT 1


Item %


Ingredient compositiona

Corn, ground yellow 55.00
Soybean meal, dehulled 37.15
Corn oil 2.50
Dicalcium phosphate 1.70
Limestone, ground 1.00
Salt, iodized .40
DL-methionine .25
Microingredientsb 1.00
Corn starch 1.00

TOTAL 100.00


Chemical compositionc

Dry matter, % 89.3
Crude protein, % 23.0
Metabolizable energy, kcal/kg 3012.0
Ca, % .95
P, % .68
Mg, % .18
Cu, mg/kgd 10.5
Mn, mg/kg 97
Zn, mg/kg 81
Fe, mg/kg 481

aAs-fed basis.

blngredients supplied per kilogram of diet: Vitamin A
palmitate, 6600 IU; vitamin D3, 2200 ICU; menadione
dimethylpyrimidinol bisulfite, 2.2 mg; riboflavin, 4.4 mg;
pantothenic acid, 13.2 mg; niacin 39.6 mg; choline chloride,
500 mg; vitamin B12, .022 mg; ethoxyquin, 125 mg;
manganese, 55 mg; iron, 80 mg; copper, 4 mg; zinc, 40 mg;
iodine, .35 mg.
cDry matter basis. Crude protein and metabolizable energy
calculated; minerals determined by analysis.
dCopper as Cu[C2H302]2 H20 added on an air dry basis at
expense of corn starch.









On day 22, chicks were individually weighed and feed

consumption for each pen replication was determined. Five

chicks from each pen were selected randomly, killed by

cervical dislocation and liver, both kidneys and right

tibia excised and frozen for subsequent mineral analysis.

Blood was collected by anterior cardiac puncture from the

remaining two chicks and plasma saved for mineral analysis.

With the exception of P, feed and tissue mineral concentra-

tions were determined by flame atomic absorption spectro-

photometry on a Perkin-Elmer Model 5000 with an AS-50

autosampler (Perkin-Elmer Corporation, 1982). Standards

were matched for macroelement and acid concentrations as

needed and standard reference material from the National

Bureau of Standards was included with samples. Phosphorus

was determined by a modified colorimetric procedure (Harris

and Popat, 1954).

Data were analyzed by least square analysis of variance

using the general linear models procedure of SAS (1982). In

the analysis of variance, pen was used as the experimental

unit for intake and feed conversion, while chick was used as

the experimental unit for gain and mineral data. Duncan's

multiple range test was used for mean separation. Plots of

residuals from the regression of raw data indicated that

liver Cu concentrations exhibited variance heterogeneity.

As a result, liver Cu concentrations were subjected to log

(Base 10) transformation prior to regression analysis.

Adequacy of regression models were confirmed by lack of









fit analysis. Regression analysis of tissue Cu was deter-

mined by the least squares method using the general linear

models procedure of SAS (1982).


Results


Analysis of chick performance (Table 3-2) indicated

that dietary Cu had no effect (P > .10) on average daily

intake, daily gain or feed conversion, which averaged

37.8 g, 24.6 g and 1.54 g/g, respectively, and were within

ranges reported as normal for chicks of this age.

Liver Cu concentrations (Table 3-3) increased from 16.7

mg/kg in chicks fed the unsupplemented basal diet to 137.5

mg/kg in those fed 300 mg/kg Cu. Regression analysis of log

transformed liver Cu concentrations indicated that this

increase was quadratic (P < .001). This is illustrated in

Figure 3-1, which shows the plot of the predicted equation

(log y = 1.20 + .0000142 X + .00000197 X2), the actual

treatment means and their associated standard errors. The

regression model accounted for 70% of the variation in

liver Cu.

Liver Mn concentrations were higher (P < .05) in chicks

fed no supplemental Cu than in those fed 100 or 200 mg/kg

Cu, but were similar to'those fed 300 mg/kg Cu. Chicks fed

300 mg/kg Cu had higher (P < .001) liver P concentrations

than those fed other dietary concentrations of Cu. Liver

Zn, Fe, Mg and Ca concentrations were not affected (P > .10)

by dietary Cu.















TABLE 3-2. EFFECT OF DIETARY COPPER CONCENTRATION ON
FEED INTAKE, GAIN AND FEED CONVERSION IN
CHICKS--EXPERIMENT 1


Added Avg. Daily Avg. Daily Feed/Unit
Cu, mg/kga Intake, g b Gain, gC Gainb



0 39.4 25.6 1.54

100 37.5 24.2 1.55

200 37.1 24.4 1.52

300 37.3 24.3 1.54

SE d 1.0 .5 .03


aBasal diet contained 10.5 mg/kg Cu,
bEach mean represents six pens.
CEach mean represents 42 chicks.
dPooled standard error.


by analysis.









TABLE 3-3. EFFECT OF DIETARY COPPER CONCENTRATION ON MINERAL COMPOSITION
OF LIVER AND KIDNEY IN CHICKS--EXPERIMENT 1


Mineral Concentration (mg/kg, dry matter basis)
Added
Cu, mg/kga
Copper Zinc Iron Manganese Magnesium Calcium Phosphorus


Liverb

0 16.7c 75.8 346 11.2d 747 314 9430c
100 16.8 76.9 356 10.3 736 315 9470
200 60.3 80.6 367 10.1 755 322 9630
300 e 137.5 76.5 355 10.6 750 302 9940
SE 9.3 1.5 23 .3 10 6 93

Kidneyb

0 14.9 73.4 300d 7.8d 827 721 12070
100 15.6 72.0 303 7.8 804 677 12390
200 16.3 74.8 280 7.3 822 705 12490
300 15.3 71.6 317 8.1 808 687 12490
SE .5 .9 8 .2 9 19 140

aBasal diet contained 10.5 mg/kg Cu, by analysis.
bEach mean represents 30 chicks.
Copper effect (P < .001).
dCopper effect (P < .05).
ePooled standard error.




























m 100- R-- 70/
a


S40


m 20


J 10V

0 1)0 260 3bO
ADDED DIETARY Cu, MG/KG



FIGURE 3-1. MEASURED AND PREDICTED COPPER CONCENTRATIONS
OF LIVER WITH RESPECT TO DIETARY COPPER INTAKE
IN CHICKS--EXPERIMENT 1









Dietary Cu had no effect (P > .10) on kidney concentra-

tions of Cu, Zn, Mg, Ca or P (Table 3-3). Kidney Fe and Mn

concentrations in chicks fed 300 mg/kg Cu were higher (P <

.05) than in those fed 200 mg/kg Cu, but were similar to

those fed 0 or 100 mg/kg Cu.

Bone ash and bone concentrations of Cu and Mg (Table

3-4) were not affected (P > .10) by dietary Cu. Bone Ca

concentrations were highest in chicks fed no supplemental

Cu, lowest in those fed 100 mg/kg Cu and intermediate in

those fed 200 or 300 mg/kg Cu (P < .001). Chicks fed 200

mg/kg Cu had lower (P < .01) bone P concentrations than

those fed 0 or 100 mg/kg Cu, but similar concentrations to

those fed 300 mg/kg Cu.

Plasma Cu concentrations (Table 3-4, Figure 3-2)

increased from 23.3 to 39.3 mg/dl as added dietary Cu

increased from 0 to 300 mg/kg Cu. Regression analysis of

plasma Cu concentrations indicated that the increase was

linear (P < .001) with the regression model accounting for

35% of the variation in plasma Cu. The plot of the pre-

dicted equation (Y = 22.4 + .0581 X), the actual treatment

means and their associated standard errors are illustrated

in Figure 3-2. Plasma Ca, P, Mg, Zn and Fe concentrations

were not affected (P > .10) by dietary Cu.


Discussion


An important requirment of the new bioassay is that

mineral levels chosen do not affect animal performance




TABLE 3-4. EFFECT OF DIETARY COPPER CONCENTRATION ON BONE ASH PERCENTAGE
AND MINERAL COMPOSITION OF BONE AND PLASMA IN CHICKS--EXPERIMENT 1


Bone Ash, % Mineral Concentration
Added Dry, Fat-
Cu, mg/kga Free Bone
Calcium Phosphorus Magnesium Copper Zinc Iron


Boneb

%, Ash Weight Basis mg/kg, Ash Weight Basis


0 38.9 39.4d 17.3e .791 3.89 ... ...
100 38.9 37.7 17.3 .915 4.74 ... ...
200 39.2 38.5 16.9 .839 4.92 ... ...
300 39.7 38.7 17.1 .867 4.24 ... ...
SEc .3 .3 .1 .040 .41

Plasmaf

mg/dl pg/dl


0 --- 8.89 7.77 1.73 23.3 160 60.7
100 --- 8.34 9.32 1.76 26.2 146 65.0
200 --- 9.49 9.06 1.80 35.8 143 47.5
300 --- 8.85 8.94 1.71 39.3 133 76.6
SE .45 .45 .08 2.6 7 7.6


a
bBasal diet contained 10.5 mg/kg
bEach mean represents 30 chicks.
dPooled standard error.
Copper effect (P < .001).
fCopper effect (P < .01).
Each mean represents 12 chicks.


Cu, by analysis.


















































0 100 200 300
ADDED DIETARY Cu, MG/KG

FIGURE 3-2. MEASURED AND PREDICTED COPPER CONCENTRATIONS
OF PLASMA WITH RESPECT TO DIETARY COPPER INTAKE
IN CHICKS--EXPERIMENT 1









(Henry et al., 1987). The highest dietary Cu concentration

used in this study is the maximum tolerable level suggested

for chicks by the National Research Council (1980). Chick

performance was not affected by dietary Cu (as acetate) up

to 300 mg/kg, indicating that those concentrations are

acceptable for use in future Cu bioassays of this type and

that the recommendations of the National Research Council

(1980) are valid.

The primary objective of this study was to determine

whether tissue Cu uptake (or its transformation) was

linearly related to dietary Cu concentration. Regression

analysis of plasma Cu concentrations indicated that Cu

uptake by plasma was linear (P < .001). However, the linear

model accounted for only 35% of the variation in plasma Cu

and the correlation coefficient (r = .59) indicated a

moderate linear association between plasma and dietary Cu.

The moderate linear association between plasma and dietary

Cu observed in this study contrasts with the very close

association (r = .99) reported by Ekperigin and Vohra

(1981b). The present study, however, differs from that of

Ekperigin and Vohra (1981b) in that feeding periods were

longer (22 vs 7 days) and Cu sources (acetate vs sulfate),

levels (0, 100, 200 and 300 vs 0, 100, 250, 500 and 1000

mg/kg) and basal diets (corn-soybean meal vs isolated

soyprotein-cornstarch) differed. These data suggest that

plasma Cu accumulation may not be a useful bioassay









criterion for determining Cu bioavailability from inorganic

sources.

Regression of liver Cu uptake with respect to dietary

Cu indicated quadratic effects in the range measured. A

graphical analysis of this response (Figure 3-1) suggests

that homeostatic mechanisms in the liver of the broiler may

be responsible for the quadratic effect observed. Liver Cu

concentrations were unchanged as dietary Cu increased from

0 (10.5 mg/kg Cu, by analysis) to 100 mg/kg added Cu, then

increased linearly from 100 to 300 mg/kg added Cu. This

ability of liver to resist changes in Cu concentrations

until dietary Cu concentrations exceed 100 mg/kg has been

observed previously (Smith, 1969; Norvell et al., 1975;

Southern and Baker, 1982) and appears to be more pronounced

in broilers fed corn-soybean meal diets. The linear

increase in liver Cu observed as dietary Cu increased from

100 to 300 mg/kg suggests that future bioassays should be

conducted within these ranges to ensure that the tissue

response falls within the linear portion of the response

curve. Use of the linear portion of the curve allows bio-

availability to be determined using slope-ratio methods.

The failure of dietary Cu to affect kidney Cu concen-

trations in this study and those of Norvell et al. (1974,

1975) and Jensen and Maurice (1979) suggest that homeostatic

mechanisms in the kidney of the broiler chick are capable of

maintaining kidney Cu concentrations over a wide range of

dietary Cu levels. In their studies, Norvell et al. (1974,









1975) fed chicks 0, 120, 240, 480 or 720 mg/kg Cu for

8 weeks and reported no increase in kidney Cu concentrations

until dietary Cu reached 720 mg/kg.

Bones were thought to be particularly responsive to

changes in dietary Cu concentrations (Underwood, 1977).

However, results of this study and that of Hedges and

Kornegay (1973) indicate that this may not be the case as

bone Cu concentrations were not affected by dietary Cu

concentrations up to 60 times the requirement. A possible

explanation for this contradiction is that studies cited by

Underwood (1977) compared deficient bones to that of Control

bones whereas in this study and that of Hedges and Kornegay

(1973) dietary Cu was well in excess of requirements.

Other than Cu, tissue minerals affected by dietary Cu

included liver Mn and P, kidney Fe and Mn and bone Ca and P.

Examination of the data, however, revealed no consistent

trends either within or across tissues and although there

were statistical differences, mineral concentrations were

within expected ranges for broiler chicks of this age

(Black, 1983; Van Ravenswaay, 1986). Data indicate that

liver Cu accumulation may be a useful bioassay criterion for

determining Cu bioavailability from inorganic Cu sources.


Summary


An experiment was conducted with 168 day-old male Cobb

feather-sexed chicks to study the relationship between high

dietary concentrations of Cu and tissue Cu accumulation.









A basal corn-soybean meal diet (10.5 mg/kg Cu) was supple-

mented with 0, 100, 200 or 300 mg/kg Cu as reagent grade

cupric acetate (Cu(C2H302)2 H20) and fed ad libitum for 22

days. On day 22, chicks were killed by cervical dislocation

and liver, both kidneys, bone and plasma samples collected

and frozen for subsequent mineral analysis. Feed intake,

gain and feed conversion were not affected (P > .10) by

dietary treatment. Copper concentrations in liver and

plasma increased (P < .001) with increasing dietary Cu,

while that of kidney and bone remained unchanged (P > .10).

Regression analysis indicated that the increase in plasma Cu

concentrations was linear (P < .001) while that of liver

was quadratic (P < .001). Other tissue minerals affected by

dietary Cu included liver Mn and P, kidney Fe and Mn and

bone Ca and P. Data indicate that liver Cu uptake may be a

useful bioassay criterion for determining Cu bioavailability

from inorganic Cu sources.















CHAPTER IV
EFFECT OF HIGH DIETARY COPPER AND AGE ON
TISSUE MINERAL COMPOSITION OF BROILER CHICKS
AS A BIOASSAY OF COPPER SOURCES--EXPERIMENT 2


Introduction


The distribution of body Cu among tissues varies with

species, age and Cu status of the animal, but it is gener-

ally higher in liver, brain, heart and kidneys (Underwood,

1977). Tissues also differ greatly in their sensitivity to

dietary Cu, with Cu concentrations in muscles, heart,

endocrine glands and kidneys being relatively independent

of dietary Cu, while that of liver, spleen, brain and bones

is very dependent (Georgievskii, 1982). Young animals have

also been shown to have higher intestinal absorption and

tissue Cu concentrations than adults of the same species.

Traditional Cu bioavailability assays are complicated

by Cu absorption and retention values that are low relative

to that of other elements. A new bioavailability assay

developed by Black et al. (1984a, b) which uses tissue

mineral accumulation during short term, high level supple-

mentation of the element as bioassay criteria, appeared to

offer some utility for Cu (Exp. 1).

The fact that absorption and tissue Cu concentrations

decrease with age suggests that age may significantly affect









a bioassay that is based on tissue accumulation. Therefore,

the present study was conducted to determine the effect of

age on tissue Cu accumulation in chicks fed high dietary

concentrations of Cu.


Materials and Methods


One hundred and ninety-two, day-old Cobb feather-sexed

male chicks were assigned randomly to pens in a thermo-

statically controlled, electrically heated Petersime battery

with raised wire floors. Chicks were maintained on a 24 h

constant-light schedule and allowed ad libitum access to

feed and tap water.

A completely randomized design with a 4 x 3 factorial

arrangement of treatments was used, with two replicate pens

of eight chicks assigned to each of 12 dietary treatments.

Dietary treatments included 0, 100, 200 or 300 mg/kg Cu as

cupric acetate [Cu(C2H302)2 H20], added to the basal diet

and fed for 1, 2, or 3 weeks. The basal diet (Table 4-1)

contained 15.2 mg/kg Cu (dry basis) by analysis and was

formulated to meet the requirements of the growing chick

(NRC, 1984).

At the end of weeks 1, 2 or 3 of the experiment, chicks

from the two replicates of each dietary treatment were

weighed individually and feed consumption was determined for

each replication. Blood was collected by anterior cardiac

puncture from two chicks per replicate and plasma saved for

mineral analysis. Chicks used to obtain blood samples and









TABLE 4-1. COMPOSITION OF BASAL DIET--EXPERIMENT 2


Item


Ingredient compositiona

Corn, ground yellow 55.00
Soybean meal, dehulled 37.15
Corn oil 2.50
Dicalcium phosphate 1.70
Limestone, ground 1.00
Salt, iodized .40
DL-methionine .25
Microingredientsb 1.00
Corn starch 1.00

TOTAL 100.00


Chemical compositionc

Dry matter, % 88.8
Crude protein, % 23.0
Metabolizable energy, kcal/kg 3012.0
Ca, % .96
P, % Total .79
Mg, % .20
Cu, mg/kgd 15.17
Mn, mg/kg 110
Zn, mg/kg 81
Fe, mg/kg 646

aAs-fed basis.

blngredients supplied per kilogram of diet: Vitamin A
palmitate, 6600 IU; vitamin D3, 2200 ICU; menadione
dimethylpyrimidinol bisulfite, 2.2 mg; riboflavin, 4.4 mg;
pantothenic acid, 13.2 mg; niacin 39.6 mg; choline chloride,
500 mg; vitamin B12, .022 mg; ethoxyquin, 125 mg;
manganese, 55 mg; iron, 80 mg; copper, 4 mg; zinc, 40 mg;
iodine, .35 mg.
CDry matter basis. Crude protein and metabolizable energy
calculated; dry matter and minerals determined by analysis.
dCopper as Cu[C2H302]2 H20 added on an air dry basis at
expense of corn starch.









the remaining six per replicate were killed by cervical

dislocation and their livers, both kidneys, pectoralis major

muscles and right tibias excised and frozen for subsequent

mineral analysis. With the exception of P, feed and tissue

mineral concentrations were determined by flame atomic

absorption spectrophotometry on a Perkin-Elmer Model 5000

with an AS-50 Autosampler (Perkin-Elmer Corporation, 1982).

Standards were matched for macroelement and acid concentra-

tions as needed and standard reference material from the

National Bureau of Standards was included with samples.

Phosphorus was determined by a modified colorimetric

procedure (Harris and Popat, 1954).

Data were analyzed by least squares analysis of

variance using the general linear models procedure of SAS

(1982). The model included main effects of age and Cu level

and their interaction. In the analysis of variance, pen was

used as the experimental unit for intake and feed conver-

sion, while chick was used as the experimental unit for gain

and mineral data. When interactions were present, inter-

action means within each factor were compared separately

using Duncan's multiple range test. Liver Cu concentrations

exhibited variance heterogeneity and were subjected to log

(Base 10) transformation prior to regression analysis.

Adequacy of regression models were verified by lack of fit

analysis. Regression analysis of tissue Cu was determined

by the least squares method using the general linear models

procedure of SAS (1982).









Results


Average daily intake, daily gain and feed conversion

of chicks fed dietary treatments for 1, 2 or 3 weeks are

presented in Table 4-2. There were significant (P < .01)

age x Cu interactions observed for intake and gain in which

response to dietary Cu varied with age. As expected, both

intake and gain increased (P < .001) with age regardless of

dietary Cu concentration. There was no age x Cu interaction

(P > .10) observed for feed conversion nor was there an

effect (P > .10) due to dietary Cu. There was, however, an

effect (P < .01) due to age with feed to gain ratio increas-

ing from 1.43 at week 1 to 1.55 by week 3.

Liver mineral composition as influenced by dietary Cu

and age is shown in Table 4-3. A significant (P < .001)

age x Cu interaction was observed for liver Cu. Liver Cu

concentrations were not affected by dietary Cu at week 1,

but increased with increasing dietary Cu at weeks 2 and 3.

Regression of log transformed liver Cu on dietary Cu by age

indicated that these increases were quadratic (P < .001).

This is illustrated in Figure 4-1 which shows plots of the

predicted equations, actual treatment means and standard

errors of those means. The regression models accounted for

66% of the variation in liver Cu at both weeks 2 and 3.

Liver Mn was not affected (P > .10) by dietary Cu, but did

increase (P < .001) with age from 8.13 mg/kg at week 1 to

10.45 mg/kg by week 3. The age x Cu interaction was








TABLE 4-2. EFFECT OF DIETARY COPPER AND AGE ON FEED INTAKE,
GAIN AND FEED CONVERSION IN CHICKS--EXPERIMENT 2


Added Copper, mg/kga Statistics
Response Age,
Weeks 0 100 200 300 Mean Effect P SEb



Intake, g/d 1 18.8C 18.0 19.1 17.6 18.4d Age .0001 .3
2 28.5 27.6 30.0 28.4 28.6 Cu .0006 .4
3 44.9 44.1 42.8 38.3 42.5 Age x Cu .0013 .6
Mean e 30.7 29.9 30.6 28.1 ---

Gain, g/d 1 13.5f 12.5 14.0 11.7 12.9g Age .0001 .3
2 19.0 19.5 21.4 20.1 20.0 Cu .0007 .4
3 h 28.5 29.0 28.2 25.2 27.7 Age x Cu .0064 .6
Mean 20.3 20.3 21.2 19.0 ---

Feed/Gain, 1 1.39c 1.47 1.36 1.51 1.43d Age .0096 .02
g/g 2 1.50 1.47 1.41 1.46 1.46 Cu .2086 .03
3 e 1.58 1.57 1.51 1.56 1.55 Age x Cu .7441 .05
Mean 1.49 1.50 1.43 1.51 ---


aBasal diet contained 15.2 mg/kg
bPooled standard error.


CEach
dEach
eEach
fEach
9Each
hEach


interaction
main effect
main effect
interaction
main effect


Cu, by analysis.


represents two pens.
for age represents eight pens.
for Cu level represents six pens.
represents 16 chicks.
for age represents 64 chicks.


main effect mean for Cu level represents 48 chicks.









TABLE 4-3. EFFECT OF DIETARY COPPER AND AGE ON THE MINERAL
COMPOSITION OF LIVER IN CHICKS--EXPERIMENT 2


Added Copper, mg/kga Statistics
Mineral Age,
Weeks
0 100 200 300 Meanb Effect P SEC



mg/kg (dry matter basis)

Cu 1 19.2d 22.7 23.8 21.7 21.9 Age .0001 3.3
2 23.3 23.4 35.1 108.2 47.5 Cu .0001 3.8
3 18.5 18.5 20.5 82.3 34.9 Age x Cu .0001 6.7
Meane 20.3 21.5 26.5 70.7 ---

Mn 1 8.32 8.60 8.49 7.09 8.13 Age .0001 .23
2 9.91 9.38 8.81 10.30 9.60 Cu .3368 .26
3 10.86 9.88 9.87 11.18 10.45 Age x Cu .0146 .45
Mean 9.70 9.29 9.06 9.52 ---

Zn 1 67.3 67.6 68.6 61.1 66.2 Age .0001 1.3
2 79.8 79.6 80.7 77.1 79.3 Cu .0293 1.6
3 82.0 72.4 78.0 73.1 76.4 Age x Cu .5205 2.7
Mean 76.4 73.2 75.8 70.4 ---

Fe 1 198 253 222 264 234 Age .0001 14
2 280 323 285 308 299 Cu .0045 16
3 318 321 306 452 349 Age x Cu .1791 28
Mean 265 299 271 342 ---









TABLE 4-3. Continued


Added Copper, mg/kga Statistics
Mineral Age,
Weeks
0 100 200 300 Meanb Effect P SEc



mg/kg (dry matter basis)

Ca 1 242d 274 247 200 241 Age .0001 6
2 311 316 309 282 304 Cu .0108 7
3 229 239 254 246 242 Age x Cu .0752 13
Meane 261 276 270 243 ---

Mg 1 659 684 665 658 667 Age .0001 9
2 770 769 764 762 766 Cu .2487 10
3 781 723 721 708 733 Age x Cu .2335 17
Mean 736 725 717 709 ---

P 1 9410 10380 10280 10180 10060 Age .0001 170
2 12330 13010 12930 12690 12740 Cu .8069 190
3 12350 11450 11330 11400 11630 Age x Cu .0525 330
Mean 11360 11610 11510 11420 ---

a Basal diet contained 15.2 mg/kg Cu, by analysis.
bEach main effect mean for age represents 48 chicks.
CPooled standard error.
dEach interaction mean represents 12 chicks.
eEach main effect mean for Cu level represents 36 chicks.













































ADDED DIETARY Cu, MG/KG


MEASURED AND PREDICTED COPPER CONCENTRATIONS OF
LIVER WITH RESPECT TO DIETARY COPPER INTAKE IN
CHICKS FED DIETS FOR 2 OR 3 WEEKS--EXPERIMENT 2


FIGURE 4-1.









significant (P < .05) for liver Mn. Liver Zn was lower

(P < .001) in birds fed 300 mg/kg Cu than in those fed 0 or

200 mg/kg Cu, but was not different from those fed 100 mg/kg

Cu and was highest (P < .05) at 2 weeks of age. Iron in

liver increased (P < .001) with age and was higher (P < .01)

at 300 mg/kg Cu than at other dietary concentrations. Liver

Ca was lower (P < .001) in birds fed 300 mg/kg Cu than in

those fed 100 or 200 mg/kg Cu, but was not different from

unsupplemented controls and was lower (P < .01) at week 2

compared to weeks 1 or 3. Liver Mg and P were highest (P <

.001) at week 2, but were not affected (P > .10) by dietary

Cu.

Table 4-4 shows the effects of dietary Cu and age on

kidney mineral composition. Kidney Cu decreased (P < .001)

with age but was not affected (P > .10) by dietary Cu.

Significant (P < .05) age x Cu interactions were observed

for kidney Mn and Fe. Kidney Zn (P < .001), Ca (P < .05)

and P (P < .001) were affected by age but not (P > .10) by

dietary Cu. Kidney Mg increased (P < .05) from 718 to 763

mg/kg as supplemental Cu increased from 0 to 300 mg/kg and

decreased (P < .05) with age.

Effects of dietary Cu and age on the mineral composi-

tion of muscle are presented in Table 4-5. Muscle Cu, Zn,

Ca, Mg and P were not affected (P > .10) by dietary Cu, but

decreased (P < .001) with age. The interaction of age x Cu

was significant (P < .05) for muscle Mn. Iron in muscle









TABLE 4-4. EFFECT OF DIETARY COPPER AND AGE ON THE MINERAL
COMPOSITION OF KIDNEY IN CHICKS--EXPERIMENT 2


Added Copper, mg/kga Statistics
Mineral Age,
Weeks
0 100 200 300 Meanb Effect P SEc



mg/kg (dry matter basis)

Cu 1 26.9d 24.6 27.9 30.0 27.4 Age .0001 .8
2 24.1 23.9 22.7 21.9 23.2 Cu .3683 1.0
3 18.7 20.3 21.8 23.5 21.1 Age x Cu .2445 1.7
Meane 23.3 22.9 24.1 25.1 ---

Mn 1 7.38 8.49 9.67 9.26 8.70 Age .0110 .19
2 7.37 8.23 7.63 8.28 7.88 Cu .0486 .22
3 8.53 8.44 8.50 7.51 8.25 Age x Cu .0021 .38
Mean 7.76 8.39 8.60 8.35 ---

Zn 1 88.4 88.2 86.3 85.2 87.0 Age .0002 1.5
2 79.9 83.6 78.6 73.2 78.8 Cu .3899 1.8
3 79.2 76.8 80.9 78.3 78.8 Age x Cu .6398 3.1
Mean 82.5 82.9 81.9 78.9 ---

Fe 1 234 249 280 236 250 Age .0001 7
2 280 276 264 276 274 Cu .6199 8
3 288 291 285 334 299 Age x Cu .0313 14
Mean 267 272 276 282 ---









TABLE 4-4. Continued


Added Copper, mg/kga Statistics
Mineral Age,
Weeks
0 100 200 300 Meanb Effect P SEc



mg/kg (dry matter basis)

Ca 1 351d 373 345 356 356 Age .0402 14
2 400 453 333 386 393 Cu .3832 17
3 337 331 354 346 342 Age x Cu .3800 29
Meane 363 386 344 362 ---

Mg 1 726 777 805 806 779 Age .0001 9
2 716 754 731 731 733 Cu .0242 11
3 712 708 726 751 724 Age x Cu .2657 19
Mean 718 746 754 763 ---

P 1 10470 10370 9960 10000 10200 Age .0001 140
2 10610 10890 10600 10810 10730 Cu .6053 160
3 11180 11030 10930 11210 11090 Age x Cu .8850 270
Mean 10760 10760 10500 10670 ---


bBasal diet contained 15.2 mg/kg Cu, by analysis.
cEach main effect mean for age represents 48 chicks.
dPooled standard error.
Each interaction mean represents 12 chicks.
e .
Each main effect mean for Cu levels represents 36 chicks.









TABLE 4-5. EFFECT OF DIETARY COPPER AND AGE ON THE MINERAL
COMPOSITION OF MUSCLE IN CHICKS--EXPERIMENT 2


Added Copper, mg/kga Statistics
Mineral Age,
Weeks
0 100 200 300 Meanb Effect P SEc



mg/kg (dry matter basis)

Cu 1 10.6d 11.5 10.1 11.5 10.9 Age .0001 .6
2 6.1 5.0 7.8 7.3 6.5 Cu .7689 .7
3 4.6 4.3 4.4 4.8 4.5 Age x Cu .7533 1.2
Meane 7.1 6.9 7.4 7.9 ---

Mn 1 1.56 1.98 1.31 2.04 1.72 Age .0001 .08
2 .84 .97 1.42 1.21 1.11 Cu .0686 .09
3 .72 .77 .76 .91 .79 Age x Cu .0332 .16
Mean 1.04 1.24 1.17 1.39 ---

Zn 1 30.8 31.3 29.9 32.7 31.2 Age .0001 .4
2 22.6 23.7 23.0 22.3 22.9 Cu .5555 .5
3 17.8 18.0 18.2 18.7 18.2 Age x Cu .5757 .9
Mean 23.7 24.3 23.7 24.6 ---

Fe 1 66.9 62.9 57.2 82.8 67.5 Age .0001 2.3
2 39.9 43.4 42.2 40.2 41.4 Cu .0397 2.6
3 30.1 31.0 29.5 37.7 32.1 Age x Cu .1134 4.6
Mean 45.6 45.8 43.0 53.6 ---









TABLE 4-5. Continued


Added Copper, mg/kga Statistics
Mineral Age,
Weeks
0 100 200 300 Meanb Effect P SEc



mg/kg (dry matter basis)

Ca 1 194d 216 229 233 218 Age .0001 10
2 164 149 198 192 176 Cu .4578 11
3 161 163 142 163 157 Age x Cu .6674 20
Meane 173 176 190 196 ---

Mg 1 1204 1228 1208 1190 1207 Age .0001 14
2 1153 1189 1188 1145 1169 Cu .9056 16
3 1052 1032 1057 1088 1058 Age x Cu .6177 27
Mean 1137 1150 1151 1141 ---

p 1 10800 11190 10820 10920 10930 Age .0001 120
2 9070 9530 8970 9090 9170 Cu .2361 140
3 8500 8660 8440 8730 8580 Age x Cu .9762 240
Mean 9460 9790 9410 9580 ---

aBasal diet contained 15.2 mg/kg Cu, by analysis.
bEach main effect mean for age represents 48 chicks.
dPooled standard error.
Each interaction mean represents 12 chicks.
eEach main effect mean for Cu level represents 36 chicks.









decreased (P < .001) with age, but was higher (P < .05) at

300 mg/kg Cu than at 0, 100 or 200 mg/kg.

Table 4-6 illustrates the effect of dietary Cu and age

on bone ash and bone mineral composition. Significant (P <

.05) age x Cu interactions were observed for bone ash and

bone P. Both main effects were also significant (P < .001)

for bone ash, but only age was significant (P < .001) for

bone P. Bone Cu was below detection limits in 1 week

chicks. There were no differences in bone Cu either as a

result of dietary Cu (P = .075) or age (P > .10). Bone Mg

was lower (P < .05) at week 2 than at weeks 1 or 3 and the

interaction of age x Cu approached significance (P = .0598).

Calcium in bone increased (P < .001) from 32.1% at week 1

to 33.4% at week 3.

Plasma mineral composition as influenced by dietary Cu

and age is presented in Table 4-7. Plasma Cu was not

affected (P > .10) by dietary Cu, but there was a trend

towards reduction in plasma Cu (P = .0725) with advanced

age. Plasma Zn was not affected (P > .10) by age or dietary

Cu. A significant (P < .01) age x Cu interaction was

observed for plasma Ca. Magnesium in plasma decreased (P <

.001) from 2.82 mg/dl at week 1 to 2.21 mg/dl at week 3, but

was not affected (P > .10) by dietary Cu. Plasma P was not

affected (P = .0926) by dietary Cu, but increased (P < .05)

from 5.79 mg/dl at week 1 to 6.33 mg/dl by week 3.












TABLE 4-6. EFFECT OF DIETARY COPPER AND AGE ON BONE ASH PERCENTAGE
AND MINERAL COMPOSITION OF BONE IN CHICKS--EXPERIMENT 2


Bone
Compo-
sition


Age,
Weeks


Added Copper, mg/kga


0 100 200 300 Meanb


Statistics


Effect


P SEC


Ash, %d 1 39.8 38.6 40.3 38.1 39.2 Age .0001 .3
2 41.5 41.0 44.6 42.5 42.4 Cu .0001 .3
3 43.9 43.6 44.6 43.5 43.9 Age x Cu .0448 .5
Meane 41.7 41.1 43.1 41.4 ---

Cu, 1 --- ... .. --- --- Age .8539 1.0
mg/kgf 2 13.6 11.5 7.2 6.2 9.6 Cu .0754 1.4
3 8.7 11.3 7.6 9.9 9.4 Age x Cu .1868 1.8
Mean 11.1 11.4 7.4 8.0 ---

Mg, % 1 .851 .887 .823 .905 .866 Age .0243 .010
2 .858 .820 .823 .804 .826 Cu .1659 .012
3 .824 .855 .833 .877 .847 Age x Cu .0598 .020
Mean .844 .854 .826 .862 ---












TABLE 4-6. Continued


Added Copper, mg/kga Statistics
Bone Age,
Compo- Weeks
sition 0 100 200 300 Meanb Effect P SEC


Ca, %f 1 31.7 32.3 31.9 32.3 32.1 Age .0001 .2
2 32.7 32.2 31.5 31.5 32.0 Cu .1639 .2
3 33.3 33.9 33.2 33.2 33.4 Age x Cu .2091 .3
Meane 32.6 32.8 32.2 32.4 ---

P, %f 1 16.7 16.4 16.0 15.8 16.2 Age .0001 .1
2 16.3 15.9 16.5 16.2 16.2 Cu .1573 .1
3 16.9 17.1 16.9 17.1 17.0 Age x Cu .0064 .2
Mean 16.6 16.5 16.5 16.3 ---

aBasal diet contained 15.2 mg/kg Cu, by analysis.
bEach main effect mean for age represents 48 chicks.
cPooled standard error.
dEach interaction mean represents 12 chicks and is expressed as a percent of
dry, fat-free bone.
eEach main effect mean for Cu level represents 36 chicks.
fEach interaction mean represents 12 chicks and is expressed on an ash weight
basis.
gCopper concentrations below detection limits.












TABLE 4-7. EFFECT OF DIETARY COPPER AND AGE ON THE MINERAL
COMPOSITION OF PLASMA IN CHICKS--EXPERIMENT 2


Added Copper, mg/kga Statistics
Mineral Age,
Weeks
0 100 200 300 Mean b Effect P SEc



Cu, 1 80.5 74.5 77.5 83.5 79.0 Age .0725 2.7
wg/dld 2 80.5 74.5 76.5 67.0 74.6 Cu .5764 3.1
3 73.0 69.5 68.0 66.0 69.1 Age x Cu .7303 5.5
Meane 78.0 72.8 74.0 72.2 ---

Zn, 1 302 299 285 330 304 Age .1303 8
Pg/dld 2 313 343 337 314 327 Cu .6495 9
3 339 335 337 284 323 Age x Cu .0994 16
Mean 318 326 319 309 ---

Ca, d 1 12.7 11.9 12.8 12.4 12.4 Age .0001 .1
mg/dld 2 15.2 14.1 13.7 14.3 14.3 Cu .0005 .1
3 13.8 13.2 12.7 11.7 12.9 Age x Cu .0055 .2
Mean 13.9 13.0 13.1 12.8 ---













TABLE 4-7. Continued


Added Copper, mg/kga Statistics
Mineral Age,
Weeks
0 100 200 300 Meanb Effect P SEc



Mg, d 1 3.10 2.58 2.96 2.66 2.82 Age .0002 .08
mg/dl 2 2.46 2.33 2.12 2.35 2.32 Cu .3124 .09
3 2.23 2.19 2.21 2.21 2.21 Age x Cu .4244 .15
Meane 2.60 2.37 2.43 2.41 ---

P'/d 1 5.50 5.21 6.36 6.07 5.79 Age .0459 .14
mg/dl 2 6.00 5.93 6.07 6.86 6.21 Cu .0926 .17
3 6.79 6.14 6.07 6.36 6.33 Age x Cu .1434 .29
Mean 6.10 5.76 6.17 6.43 ---

aBasal diet contained 15.2 mg/kg Cu, by analysis.
bEach main effect mean for age represents eight chicks.
cooled standard error.
dEach interaction mean represents two chicks.
eEach main effect mean for Cu level represents six chicks.









Discussion


The reduction in intake and gain observed in chicks fed

300 mg/kg Cu for 3 weeks, while contrasting with Exp. 1, was

not unexpected. The National Research Council (1980) lists

300 mg/kg Cu as the maximum tolerable level for chicks;

however, it is well established that dietary concentrations

of Cu in excess of 250 mg/kg often result in reduced feed

intake and growth of chicks (Smith, 1969; Jensen and

Maurice, 1979; Robbins and Baker, 1980a, b; Christmas and

Harms, 1984). This reduction in performance has been

partially attributed to a deficiency of sulfur-amino acids,

as Robbins and Baker (1980a, b) have demonstrated that

chicks fed high dietary levels of Cu (> 250 mg/kg) have an

increased sulfur-amino acid requirement. Differences

between this study and Exp. 1 may well be due to sulfur-

amino acid content of the diets, as dietary ingredients were

not identical and although both basal diets contained the

same calculated sulfur-amino acid content actual analytical

values may have been different. Performance data suggest

that sulfur-amino acid content of diets need to be increased

in future bioassays of this type.

Of tissues evaluated, only liver proved to be sensitive

to dietary Cu. The sensitivity of liver to dietary Cu is

not surprising as liver is known to be the main storage

organ of the body (Underwood, 1977). As illustrated in

Figure 4-1, liver Cu remained relatively constant until









dietary Cu exceeded 100 mg/kg then increased with increasing

dietary Cu at weeks 2 and 3. This threshold effect observed

previously (Smith, 1969; Norvell et al., 1975; Southern and

Baker, 1982; Exp. 1) is not unique to the chick, similar

effects having been observed in rats and pigs (Milne and

Weswig, 1968). The quadratic effect observed at weeks 2

and 3 in this study and in a previous experiment (Exp. 1)

indicate that liver Cu uptake cannot be assumed to be linear

down to the zero added Cu level (15.2 mg/kg). This suggests

that future bioassays should be conducted at dietary levels

that will ensure that the tissue response falls within the

linear portion of the curve. Use of the linear portion of

the curve allows bioavailability to be estimated using the

slope ratio technique and avoids the use of a nonlinear

model with its inherent complexities. The 2 week bioassay

appeared to be the more sensitive of the two assays;

however, both regression models accounted for 66% of the

variation in liver Cu.

The lack of sensitivity to dietary Cu exhibited by

kidney and muscle over the range measured is compatible with

previous reports (Norvell et al., 1975; Jensen and Maurice,

1979; Exp. 1) and is in concurrence with the observations of

Georgievskii (1982) that the Cu content of skeletal muscle

and kidney is relatively independent of the dietary Cu

concentration. In contrast, bones were thought to be

particularly responsive to changes in dietary Cu (Underwood,

1977). Results of this and previous studies (Hedges and









Kornegay, 1973; Exp. 1) indicate that this may not be the

case. No consistent trend is evident in response of plasma

to Cu supplementation. In the present study and that of

Jensen and Maurice (1979), dietary Cu concentrations up to

500 mg/kg had no effect on plasma Cu; however, in other

studies (Ekperigin and Vohra, 1981b; Exp. 1) similar dietary

Cu concentrations resulted in increased plasma Cu

concentrations.

The effect of age on tissue Cu concentrations appear to

be related to sensitivity of the tissues to dietary Cu. In

kidney and muscle, tissues least sensitive to dietary Cu,

Cu concentrations declined with age, while in plasma and

bone, tissues thought to be relatively sensitive to dietary

Cu, Cu concentrations were unaffected by age. In tissues

not sensitive to dietary Cu, decreasing Cu concentrations

with age may result from dilution as a consequence of tissue

growth. With the exception of muscle in which all minerals

including Cu decreased with age, no trends were apparent in

response of tissue minerals other than Cu to advancing age.

The reason for the variable response to advancing age is not

known.

The response of tissue minerals other than Cu to

dietary Cu concentrations, observed in the present study, is

similar to that observed in Exp. 1, in that examination of

data revealed no consistent trends either within or across

tissues. Variability in the response of tissue minerals to

dietary Cu is further demonstrated by the fact that although









tissues affected were essentially identical in both studies,

different minerals were affected. Variability of this

magnitude makes it difficult to attach biological signifi-

cance to statistical differences.

Results of the present study suggest that liver Cu

accumulation may be a useful bioassay criterion for deter-

mining Cu bioavailability and that 2 weeks is the minimum

time required for such a bioassay.


Summary


An experiment was conducted with 192 day-old male Cobb

feather-sexed chicks to study the relationship between high

dietary concentrations of Cu and tissue Cu accumulation and

to assess the influence or age on such a relationship.

Chicks were allotted randomly to a 4 x 3 factorial arrange-

ment of treatments which included a corn-soybean meal basal

diet (15.2 mg/kg Cu, DM) supplemented with 0, 100, 200 or

300 mg/kg Cu as reagent grade cupric acetate [Cu(C2H302)2

H20] and fed for 1, 2 or 3 weeks. There were two replicates

per treatment with eight chicks per replication. Chicks

were housed in a Petersime battery and given ad libitum

access to feed and tap water. At the end of each time

period, chicks were killed by cervical dislocation and

muscle, liver, both kidneys, bone and plasma samples

collected and frozen for subsequent mineral analysis.

Significant (P < .01) age x Cu interactions were observed

for intake and gain. This resulted from reductions in









intake and gain in chicks fed 300 mg/kg Cu for 3 weeks.

Feed to gain ratio was not affected (P > .10) by dietary Cu,

but did increase (P < .01) with age. Liver Cu concentra-

tions were not affected (P > .10) by dietary Cu at week 1,

but increased (P < .001) with increasing dietary Cu at weeks

2 and 3. Regression of log transformed liver Cu on dietary

Cu by age indicated that this increase was quadratic (P <

.001) in nature. Regression models accounted for 66% of the

variation in liver Cu in both weeks. Copper concentrations

in tissues other than liver were not affected (P > .10) by

dietary Cu. Other tissue minerals affected by dietary Cu

included liver Zn, Fe and Ca (P < .05), kidney Mn and Mg

(P < .05), muscle Fe (P < .05) and plasma Ca (P < .001).

With the exception of muscle in which all minerals decreased

(P < .001) with age, tissue mineral response to age varied

with tissue and mineral within tissue. Data indicated that

liver Cu accumulation may be a useful bioassay criterion for

determining Cu bioavailability and that 2 weeks is the

minimum time required for such an assay in chicks.















CHAPTER V
BIOLOGICAL AVAILABILITY OF COPPER FROM
INORGANIC SOURCES FED AT HIGH DIETARY
LEVELS TO BROILER CHICKS--EXPERIMENT 3


Introduction


The essentiality of Cu in animal nutrition and manifes-

tations of Cu deficiency are well documented (Underwood,

1977). The requirement for Cu is relatively small and, as

such, economic considerations concerning choice of a feed

grade supplement may not be critical. However, the chance

of a Cu deficiency occurring with resultant economic losses

requires consideration of the biological availability of

these sources (McNaughton et al., 1974). The biological

availability of Cu sources is also critical in the selection

of a source for use as a growth promotant or antimicrobial

since dietary levels used approach the maximum tolerable

level suggested for chicks and the chance of Cu toxicosis

occurring is greatly enhanced.

Attempts to determine Cu availability from inorganic

sources, using traditional methods have met with limited

success (Miller, 1983). Bioassays are complicated by Cu

absorption and retention values that are low relative to

that of other elements and by interactions between Cu and

other dietary constituents.









Results of previous experiments (Exp. 1 and 2)

indicated that a new bioassay technique developed by Black

et al. (1984a, b) may prove useful in determining Cu

bioavailability from inorganic sources.

This experiment was conducted to determine if the new

bioassay technique could be used successfully to determine

Cu availability from inorganic feed grade sources of Cu.


Materials and Methods


Two hundred and eight, day-old Cobb feather-sexed male

chicks were assigned randomly to pens in a thermostatically

controlled electrically heated Petersime battery with raised

wire floors. Chicks were maintained on a 24 h constant-

light schedule and allowed ad libitum access to feed and tap

water.

A completely randomized design was used with two repli-

cate pens of eight chicks assigned to each of 13 dietary

treatments. Dietary treatments included, the unsupplemented

basal (zero added Cu) and the basal supplemented with 150,

300 or 450 mg/kg Cu as either reagent grade acetate (31.8%

Cu), feed grade oxide (74.1% Cu), feed grade carbonate

(54.6% Cu) or feed grade sulfate (25.1% Cu). The basal diet

(Table 5-1) contained 11.1 mg/kg Cu (dry basis) by analysis,

and was formulated to meet requirements of the growing chick

(National Research Council, 1984). Final dietary Cu concen-

trations were confirmed by chemical analysis. Relative

solubilities (Watson et al., 1970), magnetic susceptibility









TABLE 5-1. COMPOSITION OF BASAL DIET--EXPERIMENT 3



Item %


Ingredient compositiona

Corn, ground yellow 55.00
Soybean meal, dehulled 37.15
Corn oil 2.50
Dicalcium phosphate 1.70
Limestone, ground 1.00
Salt, iodized .40
DL-methionite .45
Microingredientsb 1.00
Corn starch .80

TOTAL 100.00


Chemical compositionc

Dry matter, % 88.3
Crude protein, % 23.1
Metabolizable energy, kcal/kg 3012.0
Ca, % .98
P, % Total .80
Mg, % .21
Cu, mg/kgd 11.1
Mn, mg/kg 92
Zn, mg/kg 82
Fe, mg/kg 565

aAs-fed basis.

blngredients supplied per kilogram of diet: Vitamin A
palmitate, 6600 IU; vitamin D3, 2200 ICU; menadione
dimethylpyrimidinol bisulfite, 2.2 mg; riboflavin, 4.4 mg;
pantothenic acid, 13.2 mg; niacin 39.6 mg; choline
chloride, 500 mg; vitamin B12, .022 mg; ethoxyquin, 125 mg;
manganese, 55 mg; iron, 80 mg; copper, 4 mg; zinc, 40 mg;
iodine, .35 mg.
cDry matter basis. Crude protein and metabolizable energy
calculated; minerals determined by analysis.
dCopper as Cu[C2H30212 H20 added on an air dry basis at
expense of corn starch.









(Watson et al., 1971), chemical and physical characteristics

of Cu sources were determined and X-ray diffraction patterns

were interpreted (Table 5-2).

On day 21, chicks were weighed individually and feed

consumption for each replicate pen determined. Blood was

collected by anterior cardiac puncture from three chicks per

replicate and plasma saved for mineral analysis. Chicks

used to obtain blood samples and the remaining five per pen

were killed by cervical dislocation and their liver, both

kidneys and right tibia excised and frozen for subsequent

mineral analysis. Copper, Mn, Zn, Fe, Ca and Mg concentra-

tions in feed, tissue and Cu sources were determined by

flame atomic absorption spectrophotometry on a Perkin-Elmer

Model 5000 with an AS-50 Autosampler (Perkin-Elmer

Corporation, 1982). Standards were matched for macroelement

and acid concentrations as needed and standard reference

material from the National Bureau of Standards was included

with samples. Phosphorus was determined by a modified

colorimeteric procedure (Harris and Popat, 1954).

Data were analyzed by least squares analysis of

variance using the general linear models procedure of SAS

(1982). The model included main effects of source, Cu level

and their interaction. In the analysis of variance, pen was

used as the experimental unit for intake and feed conver-

sion, while chick was used as the experimental unit for gain

and mineral data. When interactions were present, inter-

action means within each factor were compared separately