RELATIVE BIOAVAILABILITY OF DIFFERENT

ORGANIC AND INORGANIC ZINC AND COPPER

SOURCES IN RUMINANTS AND RATS

By

LUIS X. ROJAS

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

1994

To God.

To my parents, Aquiles J. and Teresita and my brother,

Aquiles .
To my future wife, Marcia M.
To all future graduate students so they also get the

chance I got .

ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude and
appreciation to all those who collaborated in the presentation
of this dissertation.

Special appreciation is due to his advisor and chairman
of his supervisory committee, Dr. Lee R. McDowell, for his
guidance and friendship throughout all the stages of his
graduate work. Acknowledgements are also extended to Drs . D.
B. Bates, J. H. Conrad, R. J. Cousins and F. G. Martin,
members of his supervisory committee, for giving their
valuable time and knowledge toward the completion of this
research. Special recognition is due to Dr. Robert J. Cousins
for his assistance with the third part of the research and to
Dr. Frank G. Martin for his assistance in the statistical
analysis .

The author is especially grateful to his parents Dr. and
Mrs. Aquiles and Teresita Rojas for their love, encouragement
and financial support during this academic endeavor. Also,
the author wishes to recognize the Organization of American
States for their financial support.

He is deeply grateful to Mrs. Nancy Wilkinson at the
Animal Nutrition Laboratory for her guidance, assistance and
friendship in all phases of the research. Sincere

iii

appreciation is extended to Dr. Robert J. Cousins and Mrs.
Linda Ambrose for allowing the author to work in their
laboratory for analysis of metallothionein, and to the rest of
the staff for their friendship.

Special recognition is made to the staff of the Animal
Science Department and particularly Jack Stokes, Paul Dickson,
and Larry Eubanks for their help with the care and slaughter
of the animals. He also wishes to thank the faculty, staff,
and fellow graduate students of the Animal Science Department
who were always available for assistance and support.

Acknowledgement is made to Dr. Bruce Johnson and Zinpro
Corporation, Edina, Minnesota, for their generosity in
providing financial support and zinc sources for this
research.

Finally, the author wishes to recognize Ms. Marcia
Gallardo for her love and understanding during the completion
of this task.

IV

TABLE OF CONTENTS

ACKNOWLEDGMENTS iii

LIST OF TABLES vii

LIST OF FIGURES ix

ABSTRACT xi

CHAPTER 1

INTRODUCTION 1

CHAPTER 2

LITERATURE REVIEW 5

Definitions of Trace Mineral Sources 5

Trace Mineral Bioavailability 6

Factors that Affect Trace Mineral

Bioavailability 7

Assessment of Trace Element Bioavailability . 8
Comparisons of Organic and Inorganic Trace Element

Sources 9

Zinc 9

Absorption 11

Transport and Tissue Uptake 11

Bioavailability of Sources 12

Copper 14

Absorption 15

Transport and Tissue Uptake 17

Bioavailability of Sources 18

Bioavailability of Other Trace Mineral

Complexes 19

CHAPTER 3

RELATIVE BIOAVAILABILITY OF ZINC METHIONINE AND TWO

INORGANIC ZINC SOURCES FED TO CATTLE 22

Introduction 22

Materials and Methods 23

Results 26

Discussion 31

Implications 32

Summary and Conclusions 32

v

CHAPTER 4

RELATIVE BIOAVAILABILITY OF TWO ORGANIC AND TWO INORGANIC

ZINC SOURCES FED TO SHEEP 3 4

Introduction 34

Materials and Methods 3 5

Results 37

Discussion 41

Implications 46

Summary and Conclusions 46

CHAPTER 5

DEVELOPMENT OF ACUTE COPPER POISONING IN SHEEP FED ORGANIC

OR INORGANIC COPPER 48

Introduction 48

Materials and Methods 50

Results 51

Discussion 59

Implications 61

Summary and Conclusions 61

CHAPTER 6

INTERACTION OF DIFFERENT ORGANIC AND INORGANIC ZINC AND

COPPER SOURCES FED TO RATS 63

Introduction 63

Materials and Methods 64

Results 66

Discussion 74

Implications 77

Summary and Conclusions 77

CHAPTER 7

GENERAL SUMMARY AND CONCLUSIONS 7 9

APPENDIX 84

REFERENCE LIST 86

BIOGRAPHICAL SKETCH 9 8

VI

LIST OF TABLES

Table page

TABLE 3-1. Composition of concentrate diet offered to

cattle (as fed) 24

TABLE 3-2. Mean Zn concentrations in tissues of cattle

supplemented with three sources of Zn 29

TABLE 3-3. Mean Cu levels in tissues of cattle

supplemented with three sources of Zn 3 0

TABLE 3-4. Mean metallothionein levels in tissues of

cattle supplemented with three sources of Zn . . . 30

TABLE 4-1. Composition of basal diet offered to sheep

(as fed) 36

TABLE 4-2 . Mean Zn concentrations in tissues of sheep

supplemented with four sources of Zn 40

TABLE 4-3. Mean metallothionein content of tissues of

sheep supplemented with four sources of Zn . . . . 40

TABLE 4-4. Mean Cu levels in tissues of sheep

supplemented with four sources of Zn 41

TABLE 5-1. Composition of basal diet offered to sheep

(as fed) 50

TABLE 5-1. Liver and kidney Cu concentrations of sheep

exposed to high Cu levels 57

TABLE 5-2 . Liver and kidney Zn concentrations of sheep

exposed to high Cu levels 58

TABLE 6-1. Composition of purified diet fed to rats

(As-fed) 65

TABLE 6-2. Mean plasma Zn and Cu concentrations for
rats supplemented with different sources of Zn and
Cu 67

VII

TABLE 6-3 . Mean tissue Zn concentrations for rats

supplemented with different sources of Zn and Cu . 68

TABLE 6-4. Mean tissue Cu concentrations for rats

supplemented with different sources of Zn and Cu . 69

TABLE 6-5. Mean tissue metallothionein concentrations
for rats supplemented with different sources of Zn
and Cu 7 0

TABLE 6-6. Mean plasma Zn and Cu concentrations for
rats supplemented with different sources of Zn and
Cu for 4 wks and then depleted for 1 wk 71

TABLE 6-7. Mean tissue Zn concentrations for rats
supplemented with different sources of Zn and Cu
for 4 wks and depleted for 1 wk 71

TABLE 6-8. Mean tissue Cu concentrations for rats
supplemented with different sources of Zn and Cu
for 4 wks and then depleted for 1 wk 73

TABLE 6-9. Mean tissue metallothionein concentrations
for rats supplemented with different sources of Zn
and Cu for 4 wks and then depleted for 1 wk ... 74

TABLE A-l. AIN-7 6A mineral mix without added Zn or Cu

(As-fed) 84

TABLE A-2. AIN-76A vitamin mix (As-fed) 85

v 1 i i

LIST OF FIGURES

Figure page

FIGURE 3-1. Mean serum Zn for cattle supplemented with

different sources of Zn 28

FIGURE 3-2. Mean erythrocyte Zn for cattle supplemented

with different sources of Zn 28

FIGURE 3-3. Mean serum Cu for cattle supplemented with

different sources of Zn 29

FIGURE 4-1. Mean serum Zn levels for sheep supplemented

with different sources of Zn 39

FIGURE 4-2 . Mean serum Cu levels for sheep supplemented

with different sources of Zn 39

FIGURE 5-1. Serum Cu concentrations for sheep
supplemented with toxic levels of CuS04 (77 and 288)
and CuLys (1 and 46) 53

FIGURE 5-2. Serum Zn concentrations for sheep
supplemented with toxic levels of CuS04 (77 and 288)
and CuLys (1 and 46) 53

FIGURE 5-3. Blood hematocrit for sheep supplemented
with toxic levels of CuS04 (77 and 288) and CuLys (1
and 46) 54

FIGURE 5-4. Serum creatine kinase concentrations for
sheep supplemented with toxic levels of CuS04 (77
and 288) and CuLys (1 and 46) 54

FIGURE 5-5. Serum x-glutamyltransf erase levels for
sheep supplemented with toxic levels of CuS04 (77
and 288) and CuLys (1 and 46) 56

FIGURE 5-6. Serum aspartate amino transferase levels
for sheep supplemented with toxic levels of CuS04
(77 and 288) and CuLys (1 and 46) 56

IX

FIGURE 6-1 A+B. Mean bone (dry, fat free basis) Zn
concentrations for rats supplemented with different
sources. A) (Left) Different Cu sources when
supplementing different Zn sources. B) (Right)
Different Zn sources when supplementing different
Cu sources 69

FIGURE 6-2 A+B. Mean kidney (dry basis) Zn
concentrations for rats supplemented with different
sources. A) (Left) Different Cu sources when
supplementing different Zn sources. B) (Right)
Different Zn sources when supplementing different
Cu sources 73

x

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

RELATIVE BIOAVAILABILITY OF DIFFERENT ORGANIC AND INORGANIC
ZINC AND COPPER SOURCES IN RUMINANTS AND RATS

By

LUIS X. ROJAS
April 1994

Chairperson: Dr. L. R. McDowell
Major Department: Animal Science

Four experiments (EXP) were conducted to compare the

bioavailability (BAV) of amino acid complexed (Zn lysine,

ZnLys ; Cu lysine, CuLys; Zn methionine, ZnMet ) and inorganic

sources of Zn and Cu by determining Zn, Cu and metallothionein

(MT) concentrations of various fluids and tissues. In EXP 1,
cattle were given supplemental (SUP) ZnMet, ZnS04, and ZnO

(3 60 mg Zn/d) for 4 wks, withdrawn for 4 wks and resumed for
another 4 wks. No treatment (T) differences were determined
under those conditions for Zn and Cu in fluids and tissues,
and MT in tissues. In EXP 2, wethers were given SUP ZnMet,
ZnLys, ZnS04, and ZnO (360 mg Zn/kg) for 3 wks, withdrawn for
4 wks and resumed for another wk . By d 49 serum Zn had
increased less for controls than most T, and by d 55 had
increased more for ZnLys than most T. The ZnLys T had the
highest Zn and MT in kidney, liver and pancreas. Both ZnS04

xi

and ZnMet had higher liver Zn than controls. Muscle Cu was
highest for controls. For EXP 2, organic Zn sources were
equally or more BAV than the best inorganic source. In EXP 3,
four sheep were administered 250 mg of SUP Cu from either
CuLys or CuS04 . One of the CuS04 treated sheep was very
sensitive to Cu and the other was not affected by Cu excess.
Myodegradation was present in one animal from each T prior to
death. For EXP 3, the development of toxicity was not
affected by source. In EXP 4, 63 rats were given ZnMet,
ZnLys, or ZnS04 (30 mg Zn/kg) and CuLys, CuS04, or CuO (6 mg
Cu/kg) in a 3 x 3 factorial EXP. After 4 wk, four rats from
each T were sacrificed and the remaining rats fed a non-
supplemented diet for 1 wk . Plasma Cu was lower for animals
supplemented with CuO than CuS04 and CuLys. Bone Zn was
higher for CuLys than CuO. The CuO T was lowest in BAV and
kidney. Rats supplemented with CuS04 had higher muscle Cu
than with CuLys. After depletion, plasma Cu was lower for CuO
than CuLys, kidney Zn lower for CuS04 than for CuO which in
turn had the lowest liver Cu . Amino acid complexed minerals
were highly available. They were equal to and in some cases
higher in BAV than the sulfate form and considerably more
available in most cases than the oxide forms.

XI 1

CHAPTER 1
INTRODUCTION

The benefits of mineral supplementation to animals have
been known for a long time. In places where the fertility of
the soils and nutritional quality of the feed is unknown or
questionable, the use of mineral supplementation can provide
security against deficiencies. The vast majority of the
research to determine biological availability of minerals has
been conducted with supplemental inorganic sources and with
foodstuffs rather than with supplemental organic sources.

There are many methods to provide mineral supplementation
which can be grouped into direct and indirect (McDowell,
1992) . Indirect supplementation methods include the
administration of minerals to the soils in the form of
fertilizer or by changing the soil environment (e.g., pH,
moisture, etc.) which may increase the availability of some
minerals, or may encourage the growth of a specific pasture
species which may contain more of the required minerals.
Direct methods of supplementation include adding the
supplemental minerals to the feed or water, or directly

2
injecting or placing the mineral inside the animal's body
(McDowell, 1992) .

Mineral supplements originate from different sources,
with the most commonly used sources being inorganic in nature.
It is known that different forms of inorganic minerals may be
absorbed with varying efficiency.

Organic minerals are those that have an organic molecule
(e.g., amino acid, carbohydrate, protein) attached to it . A
chelated mineral is a compound which contains the metal bound
to a synthetic molecule such as ethylenediaminetetraacetic
acid (EDTA) . These chelates are usually chemically stable and
water soluble; however, the ligand is not biodegradable and,
therefore, the mineral is not always bioavailable to the
animal even after it is absorbed. In contrast, a metal-amino
acid complex is the product resulting from complexing a
soluble metal salt with an amino acid. The ligand formed in
this complex is biodegradable and, therefore, the mineral may
be more bioavailable.

Several products offering minerals in chelated form or
amino acid complexes are available for mineral
supplementation. Although considerable research has been done
concerning the performance benefits, there have been fewer
comparisons of minerals as amino acid complexes compared to
their inorganic counterparts.

Some trials have determined ruminal degradation of
different amino acid complexed minerals. There is, however,

3
a need to investigate the effect of these different sources on
increasing concentrations of these minerals in the various
fluids and tissues of the animal's body.

Zinc methionine, for example, has been determined to
bypass ruminal degradation (Heinrichs and Conrad, 1983).
Furthermore, since Zn is bound to methionine, it does not
combine with any other substrate which may make it unavailable
in the lumen of the animal and therefore it is ready for
absorption as soon as it enters the small intestine. On the
other hand, Spears (1989) observed that when Zn was deficient
in the diet, apparent absorption of Zn from either Zn
methionine or Zn oxide was similar. Zinc retention, however,
was higher in the lambs fed the Zn methionine, this suggests
a difference in the metabolism of these two sources following
absorption. It has been hypothesized by Spears et al . (1991)
that certain trace mineral chelates or complexes may enter
different pools in the body than the inorganic forms.

In determining which parameters to evaluate for Zn and Cu
metabolism research, it has been shown that the majority of
biologically available Zn and Cu is stored in the organs of
the body such as liver, kidney and pancreas with minor storage
in the bone, muscle, skin and hair, although the latter two
storage sites are not readily available to the animal. Blood
plasma and blood cells serve as immediate sources of stored Zn
and Cu . Furthermore, dietary Zn seems to affect the synthesis

4
of the metal inducible metalloprotein, metallothionein, in
some tissues (Blalock et al . , 1988).

The bioavailability of inorganic minerals has been
measured under different situations for various species (Fox
et al., 1981; Wedekind et al . , 1992; Sandoval, 1992) by
measuring several factors, such as tissue and fluid mineral
concentration, and weight gain. There have been conflicting
results concerning the bioavailability of the different Zn and
Cu sources .

CHAPTER 2
LITERATURE REVIEW

Definitions of Trace Mineral Sources

There are many organic mineral sources . The natural
organic mineral sources are those present in the environment.
A mineral chelate is a metal complex in which the metal atom
is held through more than one point of attachment to a ligand,
with the metal atom occupying a central position in the metal
complex (Morgan and Drew, 192 0) . Natural chelators are widely
distributed in all living systems in nature. Some examples of
mineral chelating agents include water, carbohydrates,
proteins, amino acids, lipids, and nucleic acids.

Depending on the bond strength of the resulting compound
or the ligand, it can then be classified as a chelate,
complex, proteinate, or other moiety (Nelson, 1988). A list
of these definitions is available (Kincaid, 1989) . A
proteinate is a product resulting from the chelation of a
soluble salt with amino acids or partially hydrolyzed protein.
A metal amino acid complex is a product resulting from the
reaction of a metal ion from a soluble metal salt with a known
amino acid.

6

The inorganic mineral sources are those which are not

bound to organic molecules, but to other inorganic elements

(e.g. sulfur, chloride, carbonate, oxide or the metal form

itself) .

Trace Mineral Bioavailability

Total concentration of a particular element in feed does
not reflect the actual amount that will be absorbed by the
animal . The reason is that frequently only a portion of that
element will solubilize and then only a portion of that will
be actually absorbed by the animal. Fox et al . (1981) defined
bioavailability as a quantitative measure of the utilization
of a nutrient under specified conditions necessary to support
the organism's normal structure and physiological process. It
is important to realize that just because an element is
absorbed does not necessarily indicate that it will be
utilized by the animal. The substance may not be metabolized
for body function and may be excreted immediately (Bender,
1989). O'Dell (1985), therefore, offers a simplified
definition of bioavailability which is the proportion of a
nutrient in a feedstuff that can be absorbed and utilized.

Bioavailability of a compound implies the availability of
that compound to some organism for body use (Miller, 1980) .
In trace elements, therefore, bioavailability refers to the
portion which can be utilized by the animal to fulfill the
functions for which the element is needed (Miller, 1980) .

Factors that Affect Trace Mineral Bioavailability

It is impossible to understand mineral bioavailability
without considering absorption. The events involved as a
substance goes from its root uptake and later incorporation
into the foodstuff to the actual fulfillment of a particular
physiological function within the body of an animal are
divided into three domains by Rosenberg and Solomons (1984) :
first, the luminal events which are responsible for the
preparation and delivery of the substance for enterocyte
uptake; second, the mucosal events which determine the
transfer of the nutrient through the enterocyte to the
basolateral membrane; and finally, the postabsorption events
which include the transport, delivery, usage, and ultimately
excretion of the nutrient.

The amount of a particular element available to the
animal depends on both intrinsic and extrinsic factors
(O'Dell, 1983) which have also been referred to as endogenous
and exogenous (Rosenberg and Solomons, 1984). The intrinsic
factors are physiological in nature and are much harder to
control. They include species and genotype (Kincaid et al . ,
1976a, b) , stage of production (Berg et al . , 1963), age
(Schisler and Kienholtz, 1967), physiological stress (Orr et
al., 1990), nutritional status (Stuart et al . , 1986), and
intestinal well being (Bafundo et al . , 1984). The extrinsic

8
factors are those which are present in the diet and include
actual concentration of the diet, chemical or physical form of
the element, presence of chelating agents, solubility of the
source, presence of interacting nutrients, and protein
concentration of the diet (Vohra and Kratzer, 1964; Rosenberg
and Solomons, 1984; Stuart et al . , 1986; Shafey et al . , 1991).
Overall, the extrinsic factors can be controlled more
efficiently to improve bioavailability.

The many factors which have been studied for their
possible effects on bioavailability include levels of
supplementation of an element (Ammerman and Miller, 1972),
elements supplied by different foods (O'Dell et al . , 1972),
different inorganic sources (Wedekind and Baker, 1990;
Sandoval, 1992), and organic sources (Hill et al . , 1986;
Pimentel et al . , 1991; Wedekind et al . , 1992). Also,
adsorption of minerals to macronutrients , binding of minerals
to other compounds, and oxidation/reduction reactions may take
place (van Dokkum, 1989) . There are also individual genetic
or physiological defects which can determine the absorption,
or lack thereof, for any particular nutrient (Rosenberg and
Solomons, 1984) .

Assessment of Trace Element Bioavailability

O'Dell (1983) suggested that the best way to assess
bioavailability was to compare absorption and utilization in
a feedstuff with those in a standard soluble salt of the

9
element. Fox et al . (1981) suggested two indices of response,
primary and secondary. Primary indices of response include
quantifiable levels of morphology or physiological function
that indicate health, like measures of growth (height, weight,
head circumference, etc.), skeletal development (bone size,
conformation, and mineralization) , hematopoiesis , circulatory-
function, etc. Secondary indices of response are quantifiable
responses that do not measure health status directly but must
be correlated with primary indices under defined conditions,
like whole body retention or concentrations of inorganic
elements, metabolites, enzymes, or hormones in tissue, body
fluids, or excretory products.

Comparisons of Organic and Inorganic Trace Element Sources

The use of inorganic mineral supplementation sources is
well known throughout the industry. These products are used
by many animal industries for their feed products as either
direct mineral mixtures with the feedstuff or by manufacturing
a separate mineral supplement. One of the most important
factors determining the use of any particular source is its
bioavailability within the target animal.

Zinc

The nutritional essentiality of Zn was demonstrated first
in the rat (Todd et al . , 1934). Nutritional interest in Zn
was increased when the element was found to be deficient in

10
swine diets in 1955, poultry in 1958, cattle in 1960, and
humans in 1961 (McDowell, 1992) .

Zinc is a bluish white metal with atomic number 30 and an
atomic weight of 65.37. Zinc is a divalent cation, with a
specific gravity of 7.13 g/cm at 20°C, and melting and boiling
points of 419.5 and 906°C, respectively. It is derived from
numerous compounds, but the principal mineral ore is the
sulfide sphalerite, which is the source of most metallic Zn

(NRC, 1979) .

The biochemical basis for the essentiality of Zn is not
completely understood. Zinc metalloenzymes can be found in
virtually every enzyme class (Vallee and Galdes, 1984) .
Several biological roles for Zn have been clarified, including
those related to cell replication and differentiation

(Hambridge et al . , 1986). Zinc has also been postulated to
have other roles independent of Zn metalloenzyme activity such
as gene expression (Wu and Wu, 1987), membrane structure and
function (Bettger and O'Dell, 1981), second messenger and
protective agent in molecular storage systems (Grummt et al . ,
1986), and improvement of stability of human growth hormone

(Cunningham et al . , 1991) . A great portion of the current
research on Zn metabolism is aimed at its functions in
molecular biology and nucleic acids.

11

Absorption

The specific gastrointestinal site where the majority of
Zn is absorbed has not been identified (Solomons and Cousins,
1984) . All of the sections of the small intestine may have a
functional importance in Zn absorption (Cousins and Hempe,
1990) .

Several investigators (Steel and Cousins, 1985; Hoadley
et al . , 1987) have reported that two kinetic processes are
involved in absorption, passive diffusion and carrier-mediated
components that may represent paracellular and intracellular
absorption pathways. Carrier-mediated Zn absorption may
increases during periods of low Zn intake, suggesting the
stimulation of a carrier system to absorb greater amounts of
Zn during a deficient state (Hoadley et al . , 1987) . In
contrast, the diffusion component of Zn absorption is
unaffected by Zn deficiency, and absorption via this process
is proportional to luminal Zn concentration. Metallothionein
(MT) synthesis is influenced both by dietary Zn level and by
plasma Zn concentration and can regulate the quantity of Zn
entering the body, thus playing a central role in Zn
homeostasis (Cousins and Hempe, 1990) .

Transport and Tissue Uptake

Albumin appears to be the main Zn carrier in blood with
approximately 70% of the Zn bound to it (Vikbladh, 1950).
Other Zn-containing components of plasma are oc2-macroglobulin

12
(Parisi and Vallee, 1970), transferrin (Charlwood, 1979), and
the amino acids cysteine and histidine (Morgan, 1981) . Plasma
Zn represents less than 1% of the total body content but
serves as a primary source of the element accessible to all
cells (Vallee and Falchuk, 1993). The exact mechanisms for
tissue uptake of Zn are not well known. Tissue Zn
concentration in most mammalian tissues has been reviewed
(Hambridge et al . , 1986), and tissue Zn levels were fairly
constant among species. The tissues most sensitive to excess
dietary Zn intake include liver, kidney, pancreas, small
intestine, and bone (Kincaid et al . , 1976a, b) . Concentration
changes of trace elements in tissues have been used as
indicators of Zn bioavailability in rats and sheep (Moncilovic
et al., 1975; Henry et al . , 1988).

Bioavailability of Sources

The two predominant Zn sources used by the animal feed
industry are ZnS04 (36% Zn) and ZnO (72% Zn) . It has been
suggested that the mineral source plays an important role in
the formation of unknown complexes inside the digestive tract
which in turn limit their absorption and further metabolism
(Hughes, 1984; Clydesdale, 1990), but there are many
contradicting studies as to the different effects and
bioavailabilities of different sources. Zinc as the metal,
sulfate, carbonate, oxide, and in several natural ores has
been shown to be relatively available when provided in

13
suitable physical forms (Ammerman and Miller, 1972). In
chicks, bioavailability of ZnO was determined to be 44.1% that
of ZnS04 (Wedekind and Baker, 1990). In another study
(Wedekind et al . , 1992) Zn methionine (ZnMet) was reported to
be better than both ZnS04 and ZnO.

In cattle, ZnMet is not broken down by ruminal
microorganisms (Heinrichs and Conrad, 1983) and was found to
be more bioavailable than ZnO (Chirase et al . , 1991). In
pigs, however, ZnMet was found to be of equal bioavailability
with ZnS04 (Hill et al . , 1986). In lambs, ZnO and ZnMet were
absorbed to a similar extent, but were metabolized differently
after absorption (Spears, 1989) . In a summary of ZnMet
studies by Herrick (1989), ZnMet increased gain and feed
efficiency by an average of 3.5% in feedlot cattle. Also the
addition of ZnMet to diets of lactating dairy cows has
increased milk production and reduced somatic cell counts in
milk (Herrick, 1989; Kellogg et al . , 1989).

In determining performance and mineral metabolism of
lambs, Kegley and Spears (1992) suggested ZnO and ZnS04
improved performance, but ZnMet had no effect. Chirase et al .
(1992) determined that Zn and Mn methionine improve the
recovery rates of calves stressed with infectious bovine
rhinotracheitis virus. In lambs, the retention of a Zn
proteinate source was higher than that of ZnO (Lardy et al . ,
1993) . Visual hoof score and hoof durability have also been
suggested to be affected by Zn supplement (Moore et al . , 1992;

14
Reiling et al . , 1992). Although hoof growth and wear
measurements were similar for ZnMet supplemented dairy cows
versus controls, visual hoof score (texture, heel cracks,
laminitis, ulcers, interdigital dermatitis and hoof rot)
showed improvement with ZnMet (Moore et al . , 1992). Hoofs
from Zn proteinate supplemented heifers had a higher shearing
force than those from ZnS04 supplemented animals (Reiling et
al., 1992). In young pigs, Hall et al . (1993) suggested
increased availability of ZnMet versus ZnO supplementation.
Rust and Schlegel (1993) reported no differences in steer
performance or carcass characteristics with ZnO or ZnMet
supplementation .

Copper

The nutritional essentiality of Cu was demonstrated first
by McHargue (1925) based on the wide distribution of Cu in
plant and animal tissues. Interest in Cu nutrition grew in
the 1930s when Becker et al . (1931) and Neal et al. (1931)
reported that Cu was responsible for a condition in Florida's
cattle known as "salt sickness."

Copper has an atomic number 2 9 and an atomic weight of
63.55. Copper can exist as the metallic form or in +1, +2 or
+3 valence states. The most common is the +2 state (Miller,
1979) . It tends to occur in sulfide deposits, particularly
igneous rocks, with concentrations in the continental crust of
5 0 ppm .

15
Understanding of the nutritional and metabolic roles of
Cu is based on the functions of the known Cu-enzymes and on
its role in disulfide bonding of keratin by an unknown
mechanism; however, knowledge of its biological roles remains
incomplete (Danks, 1988) . In animals there are approximately
ten proteins that are generally accepted as true cuproenzymes
(Prohaska, 1988) . In addition to the known cuproenzymes with
specific functions, there are about 12 proteins of unknown
functions that when isolated contain one or more Cu atoms
(Prohaska, 1988) . Some of the ten known cuproenzymes include:
1) tyrosinase (formation of melanin); 2) lysil oxidase
(synthesis of structural subunits of collagen and elastin) ; 3)
dopamine S-hydroxylase (adrenal synthesis of catecholamine; 4)
superoxide dismutase (immune, antioxidant function); 5)
cytochrome C oxidase (energy metabolism via oxidative
phosphorylation) (Allen and Solomons, 1984) . Copper has also
been suggested in the mineralization of growing bone, either
in a cuproenzyme with ascorbate oxidase activity, or in its
soluble ionic form (Hsieh and Hsu, 1980) . A great deal of
current research on Cu metabolism is focused on the use of Cu
to improve immune response.

Absorption

In monogastrics , Cu is absorbed from all segments of the
gastrointestinal tract including stomach and large intestine
(Mason, 1979; Davis and Mertz, 1986). The major site of Cu

16
absorption is species dependant. The duodenum, however, has
been generally accepted as the primary site for Cu absorption
in most species (O'Dell, 1990) .

The mechanism of Cu absorption is not clear, but
absorption is known to be regulated at the intestinal mucosa.
Passage of Cu across mucosal membrane and transport across
cells are concentration-dependent and saturable and uptake by
mucosal cells is not energy dependent (Crampton et al . , 1965) .
Since MT has a stronger affinity for Cu than for Zn, the
protein greatly influences Cu absorption in intestinal cells
(Cousins, 1985) . In adequate or high dietary Cu, when the
animals demand of Cu is low, Cu enters the enterocyte and
binds to MT preventing any additional uptake. With low
dietary Cu, the MT bound Cu present in the intestine would
have been released through the basolateral membrane to the
portal vein and consequently taken to the liver. Sheep,
however, are more sensitive to Cu toxicosis because of the
lack of intestinal MT synthesis (Saylor et al . , 1980).
Furthermore, Turner et al . (1987) working with everted sacks
of sheep jejunum, suggested that Cu uptake from lumen to cells
was a process neither saturable nor energy-dependant but whose
kinetics reflected that of simple diffusion. Another
important factor in Cu homeostasis in sheep is that sheep
cannot excrete high amounts of Cu in bile acids (Gooneratne et
al. , 1989) .

17
One of the most powerful antagonists of Cu absorption in
general seems to be Zn (O'Dell, 1985) . Excess dietary Zn has
been reported to aggravate the signs of low Cu status (L'Abbe
and Fischer, 1984) . In rats fed adequate Cu levels (6 mg/kg) ,
Zn dietary concentrations of 120 and 240 mg/kg depressed the
activities of important cuproenzymes such as liver superoxide
dismutase and heart cytochrome C oxidase (L'Abbe and Fischer,
1984) . This antagonistic effect appears to take place mainly
in the intestinal mucosa via MT (Cousins, 1985) .

Transport and Tissue Uptake

As with Zn, albumin appears to be the main Cu carrier in
portal blood. After passing through enterocytes, Cu is
transported through portal blood as a histidine-Cu-albumin
complex (Lau and Sarkar, 1971). There are controversial
reports as to the actual carrier of Cu in peripheral
circulation. After transport into hepatocytes, Cu is released
into the blood stream bound mostly to ceruloplasmin . Copper
seems to stay bound to ceruloplasmin through peripheral
circulation (O'Dell, 1990). Bremmer (1980), however, suggests
that the principal transport forms of Cu are its loosely bound
complexes with albumin and, to a lesser extent, to selected
amino acids which include histidine, threonine and glutamine.
Hepatic Cu is temporarily stored complexed to ceruloplasmin
and released into plasma or bile as such (Bremmer, 1980) .

18
Other suggested storage proteins in liver include superoxide
dismutase, MT, and mitochondrocuprein (Bremmer , 1980) .

Bioavailability of Sources

Supplementation of Cu as CuS04, CuC03/ CuCl2 or Cu(N03)2
resulted in similar elevations in blood and plasma Cu
concentrations in sheep (Lassiter and Bell, 1960) and cattle
(Chapman and Bell, 1963), but both Cu:0 and CuO forms were
less available (Lassiter and Bell, 1960) . In the growing
chick, Cu from Cul and Cu20 were 82 and 76%, respectively, as
available as CuS04 (McNaughton et al . , 1974) . According to Ho
et al . (1980) , amino acid complexes or organically bound forms
apparently have a higher bioavailability than inorganic Cu
sources because of their ability to prevent the occurrence of
hypocupremia in beef cattle.

Other recent studies with swine (Cromwell et al . , 1989),
chicks (Baker et al . , 1991), sheep (Pott et al . , 1992) and
cattle (Clark et al . , 1993) show that CuS04 is more available
than CuO. Baker et al . (1991), however, suggested similar
bioavailability for Cu from a Cu lysine (CuLys) complex to
that of CuS04 in chicks. Liver Cu has been reported to
increase more rapidly when cattle were supplemented a Cu
proteinate versus CuS04 (Clark et al . , 1993) . In studies with
weanling pigs (Coffey et al . , 1992; Coffey et al., 1993), it
was suggested that CuLys and CuS04 were not only similar for
many aspects of bioavailability but that CuLys was also an

19
effective growth promotant for weanling pigs. In sheep
studies (Pott et al . , 1992), Cu from CuCl2, CuS04, CuC03, and
Cu acetate sources were of similar bioavailability. Nockels
et al . (1993) suggest that CuLys is better retained than that
of CuS04 in stressed calves and that significant changes
occurred in Cu and Zn balance with supplementation and stress.
In a bioavailability study using growing cattle, Kegley
and Spears (1993) suggested that Cu from CuLys was of similar
bioavailability to that from CuS04 but these were higher than
CuO. Kincaid et al . (1986) using a diet high in Mo and S
found Cu proteinate to increase Cu status (plasma and liver
Cu) more readily than CuS04 suggesting the Cu proteinate to be
less affected by high Mo. In two separate studies, however,
Wittenberg et al . (1990) showed no difference between Cu
proteinate and CuS04 as to their effect on the Cu status of Cu
depleted steers fed diets high in Mo. Ward et al . (1993)
supplemented CuLys or CuS04 to growing steers fed a diet with
or without supplemental Mo and S. They found no difference
between CuLys and CuS04 bioavailability using growth rate,
feed intake, feed efficiency, plasma Cu, ceruloplasmin
activity, and immune response as indicators of Cu status.

Bioavailability of Other Trace Mineral Complexes

Ward et al . (1992) suggested that a mixture of Zn, Mn, Cu
and Co in amino acid complexed forms may stimulate feed intake
and growth during the initial stress period of feedlot steers

20
compared to the oxide or sulfate forms. A 75% inorganic and
25% proteinate Zn, Mn and Cu mixture provided as a dietary
supplement improved embryo and/or fetal survival, and reduced
the duration of estrus in sows compared to those supplemented
with an organic mixture (Mirando et al . , 1993) .

Spears (1991) showed, feeding a Mn deficient diet to
heifers, how Mn methionine (MnMet) supplementation was
superior to MnO in improving growth and feed efficiency.
Henry et al . (1992) suggested MnMet to be equally available to
MnS04 but more available than MnO. The relative
bioavailability of Mn proteinate was similar to that of MnS04
in chicks fed diets either devoid of or containing fiber and
phytate (Baker and Halpin, 1987) . Also in chicks, MnMet was
174% more available (based on bone Mn accumulation) than MnO
(Fly et al., 1989), and in another study (Henry et al . , 1989)
MnMet was not only more available than MnO but also than
MnS04 .

Organic iodine (ethylenediamine dihydroiodide)
supplemented mice had similar macrophage phagocytosis to NaI03
and Nal supplemented mice (Siddiqui et al . , 1993) .

In a toxicity and tissue retention study conducted with
rats, Na2Se04 was found to be more toxic to methionine
deficient rats than L-selenomethionine (SeMet) (Salbe and
Levander, 1990) . For pigs, however, SeMet was more toxic than
the inorganic form (Herigstad et al . , 1973) . Furthermore,
SeMet has been shown to protect chicks against pancreatic

21
atrophy more effectively than inorganic Se (Cantor et al . ,
1975) .

In supplementing a complexed form of Co (Co dextro lac)
to feedlot cattle, Carpenter et al . (1992) suggested no
benefits in terms of animal performance versus no
supplementation. Using growing-finishing pigs, Mooney and
Cromwell (1993) indicated that Cr picolinate resulted in
carcasses with increased percentages of muscle and decreased
percentages of fat versus controls.

CHAPTER 3

RELATIVE BIOAVAILABILITY OF ZINC METHIONINE

AND TWO INORGANIC ZINC SOURCES

FED TO CATTLE

Introduction

Several products offering minerals in chelated form or
complexed with amino acids are available for mineral
supplementation. Zinc methionine (ZnMet) can bypass rurninal
degradation (Heinrichs and Conrad, 1983) . Furthermore, it
does not combine with any other substrate which may render it
unavailable in the lumen of the animal and, therefore, it is
ready for absorption upon entering the small intestine.
Spears (1989) found that when a deficient diet was fed,
apparent absorption of Zn from ZnMet or ZnO was similar, but
Zn retention increased by ZnMet suggesting different
metabolism following absorption. Spears et al . (1991)
hypothesized that organic sources enter different body pools
than inorganic forms. The popular Zn sources used by the
animal feed industry are ZnS04 (36% Zn) and ZnO (72% Zn) ;
therefore, it is necessary to test other products against
those currently being used.

The majority of bioavailable Zn when supplemented in
relatively high levels is stored in body organs such as liver,

22

23
kidney and pancreas with minor storage in bone, muscle, skin
and hair (Ott et al . , 1966) . Blood plasma and blood cells
serve as immediate sources of stored Zn. Increasing dietary
Zn has also been shown to stimulate the production of the
protein metallothionein (MT) in some tissues (Blalock et al.,
1988) .

The present study was undertaken to compare the effect of
supplemental ZnMet , ZnS04, and ZnO on Zn, Cu and MT
concentrations in various fluids and tissues of the animal's
body.

Materials and Methods

Thirty-two yearling Limousine and Angus cross-bred
heifers ranging from 213 to 318 kg and averaging 256 ± 31
(mean ± standard error of the mean; SEM) kg were used in a 12
wk experiment . Four wks prior to the experimental period
animals were randomly assigned and housed in 4 earth pens (511
nr, eight animals per pen) for 2 wk . This provided an
adjustment and training period in which heifers received a
diet without Zn supplementation and low quality Bermuda grass
hay in order to minimize Zn stores. Subsequently, treatments
were randomly assigned to animals. The treatments consisted
of three different Zn sources to supply 360 mg/d of
supplemental Zn: ZnMet (Zinpro Corporation, Edina, MN) , ZnS04
or ZnO (Southeastern Minerals, Bainbridge, GA) and a negative
control group which received no supplemental Zn . To provide

24
the daily supplemental Zn the diets were formulated to contain
200 mg Zn/kg diet. Animals were fed individually via Calan
gates 1.8 kg of a corn-based concentrate into which the three
different Zn sources were mixed. Bermuda grass hay was
offered ad libitum. Zinc content (Dry matter basis, DMB) was
19.6 mg/kg in hay and ranged from 20 to 26 mg/kg in the
control diet (Table 3-1) . The diet was formulated to be
adequate in protein, energy, vitamins, and minerals for this
class of cattle (NRC, 1984). Heifers were given supplemental
Zn for 4 wks, depleted (not supplemented) the following 4 wks
and then supplemented for 4 wks . Protocol for animal care had
been approved by the University of Florida's Institutional
Animal Care and Use Committee. Weights were obtained monthly

TABLE 3-1. Composition of concentrate diet
offered to cattle (as fed)a.

Ingredient Percentage

Corn 84.07

Soybean meal (44% CP) 14.3 0

Dicalcium phosphate 1.05

Saltb .29

Trace mineral mix0 .26

Vitamins A and D3d .03

a Zn analysis biweekly indicated means of 248, 251 and

256 mg/kg for ZnMet, ZnS04 and ZnO, respectively. Hay

intake ranged from 2 to 5 kg (as-fed) .

b Provided 2.1 g of NaCl per kilogram of diet.

r Provided .24 mg of I (KI), .28 mg of Co (CoCOJ, 82 mg

of Fe (FeS04), 38 mg of Cu (CuCl), 48 mg of Mn "(MnS04)

and no Zn per kg of diet .

a Provided 5,000 IU of vitamin A and 500 IU of vitamin

D, per kg of diet .

25
and blood samples were obtained via jugular venipuncture on d
1 before animals were administered the concentrate and
thereafter on d 3, 14 and biweekly. To obtain serum, samples
were centrifuged at 700 x g for 25 min, supernatant decanted
and frozen until analyzed for Zn and Cu levels. Erythrocytes
were harvested from centrifuged (700 x g for 25 min) whole
blood from which plasma had been removed and had been washed
twice with 9 M cold saline solution. An erythrocyte lyase was
prepared by combining 1 ml erythrocytes with 1 . 5 ml deionized
water and frozen for storage. At the end of the experiment on
d 84, animals were stunned with a captive bolt shot and
euthanized by exsanguination .

Liver, pancreas, kidney, bone and bone marrow
(metacarpus), skin, hair, hoof, neck muscle (sterno
mandibularis) , and eye were excised and frozen for further
mineral analyses. Zinc and Cu in tissues, blood constituents
and diet samples collected were determined by air acetylene
flame atomic absorption spectrophotometry on a Perkin-Elmer
Model 5000 with AS-50.

Metallothionein was measured in liver, pancreas and
kidney by the 10QCdJ+-hemoglobin affinity assay (Eaton and Toal,
1982) . For this procedure .2 g of the tissue was homogenized
with 4 volumes of cold lOmM Tris-HCl buffer (pH 7.4), with a
Potter-Elvehjem glass-teflon tissue grinder, and centrifuged
(40,000 x g; 10 min; 4°C) . The supernatant was then heated
(100°C; 5 min) and centrifuged (10,000 x g; 5 min) . Next, 200

26
|ll of Cd solution (2 |ig Cd and .5 |iCi 109Cd per ml of 10 mM
Tris-HCl buffer, pH 7.4) were added to a 200 [ll aliquot of the
supernatant and the mixture was incubated (10 min; room
temperature) . Finally, 100 |J.l of 2% hemoglobin were added.
The sample was then heated (100°C; 2 min) and centrifuged

(10,000 x g; 5 min) . This step was then repeated and 100 (il
of the supernatant and standard solutions were placed in a
Gamma Spectrometer Model 4000 (Beckman Instruments, Inc.) to
measure 10yCd levels. These levels were then used to assess MT
concentrations .

The experiment was designed as a 4 x 4 factorial in a
completely randomized experiment. There were four Zn
treatments, three sources of Zn and one negative control, and
four levels of vitamin E. All data were analyzed using SAS

(SAS, 1988) . Repeated measures ANOVA was performed using the
general linear model (GLM) procedure on changes (increase or
decrease from d 1) in Zn and Cu concentrations in serum, and
on changes in Zn concentrations in erythrocytes. Tissue Zn
and Cu data were analyzed using GLM. In case of significance

(P < .05) in serum or tissue data, Waller-Duncan's K-ratio T
test was used for multiple comparisons.

Results

Since no zinc x vitamin E interaction was found the
statistical evaluation was based on main effects. The vitamin
E data were discussed by Njeru et al . (1993) . Weight gains

27
were not different (P > .05) among treatments. Concentrate
intakes were similar for all groups with no anorexia noted.

There were no treatment differences (P > .05) in serum Zn
content for all days of collection (Figure 3-1), with the
amount of dietary Zn not playing any role in controlling serum
Zn, since the unsupplemented controls were not different.
Surprisingly, on d 28 (beginning of depletion phase) serum Zn
in most treatments (including the control) started increasing
and upon repletion (d 56) levels started falling again. As
with serum Zn, erythrocyte Zn was not affected (P > .05) by
treatment (Figure 3-2) . Unlike serum Zn, however, Zn content
of erythrocytes dropped in all treatments with depletion and
stabilized with repletion.

Overall mean serum Cu concentrations fluctuated greatly
but tended to decrease with all Zn treatments (Figure 3-3) .
By d 56 the Cu concentration increased (from d 1) by .17 ± .07
|ig/ml (mean ± SEM) for ZnO treatment and was greater (P < .05)
than both ZnMet and control treatments which dropped by -.04
± .05 and -.05 ± .07 (ig/ml, respectively. By d 70 the Cu
concentrations for the ZnO treated sheep (-.02 ± .13) had
decreased less (P < .05) than other treatments.

There were no treatment differences (P > .05) in Zn

(Table 3-2) and Cu (Table 3-3) tissue concentrations and

liver, kidney and pancreas MT concentrations (Table 3-4) . As

with the blood data, no differences were seen among Zn sources

or even between the supplemented treatments and the

28

1.4

20

30 40 50

Days on trial

60

70

80

FIGURE 3-1. Mean serum Zn for cattle supplemented with
different sources of Zn . SEM (|ig/ml) for d 1 .20, d 3 .18, d
14 .25, d 28 .21, d 42 .21, d 56 .36, d 70 .32, d 84 .29.

2.8

FIGURE 3-2. Mean erythrocyte Zn for cattle supplemented with
different sources of Zn . SEM (|ig/ml) for d 1 .45, d 3 .43, d
14 .31, d 28 .33, d 42 .34, d 56 .33, d 70 .40, d 84 .36.

29

0 10 20 30 40 50 60 70 80

Days on trial

FIGURE 3-3. Mean serum Cu for cattle supplemented with
different sources of Zn . SEM (|ig/ml) for d 1 .20, d 3 .15, d
14 .18, d 28 .21, d 42 .24, d 56 .16, d 70 .24, d 84 .16.

TABLE 3-2. Mean Zn concentrations in tissues of cattle
supplemented with three sources of Zn (mg/kg, DMBa)

Tissue'1'

Control

ZnMet

ZnS04

ZnO

SEM

Boned

60

60

61

62

8

Bone Marrow"

143

137

133

133

22

Cornea

2.8

2.3

3.2

2.8

.97

Skin

1.9

1.5

1.8

2 .2

1.4

Hair

86

88

86

84

8

Hoof

77

74

70

81

10

Kidney

72

67

69

69

5

Liver

111

116

118

114

18

Muscled

190

179

182

192

25

Pancreas

65

66

66

62

10

a DMB= Dry matter basis, bone also fat free, cornea and skin on wet

basis .

b No difference among treatments (P > .05)

c Metacarpus .

d Sterno mandibularis .

30

TABLE 3-3. Mean Cu levels in tissues of cattle
supplemented with three sources of Zn (mg/kg, DMBa

Tissueb

Control

ZnMet

ZnS04

ZnO

SEM

Bone

6

6

5

6

1.4

Bone Marrow

28

30

28

30

7

Hair

6

6

6

6

.9

Hoof

2.2

1.9

2.0

2.2

.8

Kidney

17

17

16

16

3

Liver

172

163

215

150

56

Muscle

4

4

4

4

1

Pancreas

4

4

5

5

1

a DMB= Dry matter basis, bone also fat free basis.
b No difference among treatments (P > .05) .

TABLE 3-4. Mean metallothionein levels in tissues of
cattle supplemented with three sources of Zn (|ig MT/ga

Tissue1'

Control

ZnMet

ZnS04

ZnO

SEM

Liver

140

125

108

139

57

Kidney

68

63

63

56

19

Pancreas

43

46

29

44

18

a Jig MT/g= \ig of Metallothionein per gram of wet tissue.
b No difference among treatments (P > .05)

unsupplemented control. Copper concentrations of the tissues
did not drop in the supplemented treatments as might have been
expected when compared to controls and stayed similar
throughout treatments and within tissues.

31
Discussion

Since dietary Zn levels were relatively high this may
have accounted for lack of difference among Zn sources.
Animals were receiving up to 80 mg of Zn/d from the control
diet alone. On the other hand both the hay (19.6 mg of Zn/kg)
and the concentrate (20 to 26 mg of Zn/kg) were below the
minimum adequate level of 30 mg/kg (NRC, 1984) . Spears (1989)
showed increased retention of Zn in lambs supplemented with
ZnMet compared to ZnO . In this study no differences were
observed in serum Zn concentrations even when all the animals
were given the same amount of Zn for the 4 wks of depletion.
It would have been beneficial, however, to decrease the Zn
level in the basal diet to try to stimulate Zn mobilizing
mechanisms . There were a few unexplainable serum Cu
differences on d 56 and 70. These could be related to stress
since ceruloplasmin induction produced by stress would elevate
serum Cu levels (Cousins, 1985) .

Availability of Zn sources are in agreement with other
researchers in studies with swine (Hill et al . , 1986) and
chicks (Pimentel et . al . , 1991). These researchers indicated
no differences in availability between organic and inorganic
sources of Zn . Wedekind and Baker (1990) showed increased
bone Zn deposition in chicks fed ZnMet relative to ZnO and
ZnS04. Wedekind et al . (1992) also suggested that bone Zn
levels increased when ZnMet was used compared to ZnS04 or ZnO,
however, they did not use fat-free bone in their study. Lack

32
of a difference among sources in the present study is also in
agreement with a sheep experiment to determine if Zn from
ZnMet would influence muscle (longissimus or biceps femoris)
Zn concentrations (Medeiros et al . , 1989) . A genetic
difference might also exist between the species used by other
researchers in terms of their Zn metabolism especially between
ruminants and monogastrics . It is suggested that this trial
be conducted using similar levels of supplemental Zn in
combination with lower levels of basal dietary Zn . This kind
of study would be more expensive because it would necessitate
the use of purified or semipurified diet.

Implications

These results suggest that at adequate levels of dietary
Zn, bioavailability of supplemental Zn sources may be less
important than under conditions of limited dietary Zn or
increased supplemental Zn .

Summary and Conclusions

A 12 wk experiment was conducted to compare supplemental
ZnMet, ZnS04, and ZnO on Zn, Cu and MT concentrations in
various fluids and tissues of 32 yearling cattle.
Supplemental Zn (3 60 mg/d) was fed for 4 wks, withdrawn for 4
wks and then resumed for another 4 wks. Mineral ( Zn and Cu)
concentrations were determined in serum, liver, pancreas,
kidney, bone, bone marrow, hair, hoof and neck muscle, and Zn

33
only in erythrocytes, skin, and cornea. Metallothionien
levels were determined in liver, pancreas and kidney. There
were no treatment differences (P > .05) in serum or
erythrocyte Zn content for all days of collection. Serum Cu
concentrations tended to decrease with all treatments. There
were no treatment differences (P > .05) in Zn and Cu tissue
concentrations and liver, kidney and pancreas MT
concentrations. Tissue Cu concentrations did not drop in the
supplemented treatments when compared to controls. At
adequate levels of dietary Zn, bioavailability of supplemental
Zn sources may be less important than under conditions of
limited dietary Zn or if very high levels of supplemental Zn
are fed.

CHAPTER 4

RELATIVE BIOAVAILABILITY OF TWO ORGANIC

AND TWO INORGANIC ZINC SOURCES

FED TO SHEEP

Introduction

The use of amino acid complex minerals in mineral
supplements compared to inorganic forms is still
controversial . Limited research has been done concerning the
biological availability of organic and inorganic mineral
sources. In a trial to test bioavailability, Spears (1989)
found that when a deficient diet was fed, apparent absorption
of Zn from Zn methionine (ZnMet) or ZnO forms was similar, but
Zn retention increased with ZnMet, suggesting different
metabolism following absorption.

The majority of bioavailable Zn when supplemented in
relatively high levels is stored in body organs such as liver,
kidney and pancreas with minor storage in bone, muscle and
skin (Ott et al . , 1966) . Blood plasma serves as an immediate
source of stored Zn . Dietary Zn also stimulates production of
the protein metallothionein (MT) in some tissues (Blalock et
al. , 1988) .

The objectives of this study were to compare
bioavailability of two organic and two inorganic Zn sources in

34

35
sheep by evaluating Zn and Cu concentrations of selected
tissues and serum, and MT in kidney, pancreas and liver.

Materials and Methods

Forty crossbred wether lambs averaging 37.6 ± 3.1 kg
(mean ± SEM) were randomly assigned to one of five treatments.
The treatments consisted of four different sources to supply
3 60 mg/d of supplemental Zn : ZnMet, Zn lysine (ZnLys; Zinpro
Corporation, Edina, MN) , ZnS04 or ZnO (Southeastern Minerals,
Bainbridge, GA) and a negative control group which received no
supplemental Zn . The basal diet contained from 16 to 20 mg/kg
Zn (Table 4-1) . The diet was formulated to be adequate in
protein, energy, vitamins, and minerals for this class of
sheep (NRC, 1985) .

Lambs were housed in individual wooden pens (1.4 irr) with
expanded metal floors in an open sided barn. Feed intake was
restricted to 1000 g/hd daily (as-fed basis) with tap water
available ad libitum. The protocol for animal care had been
approved by the University of Florida's Institutional Animal
Care and Use Committee.

Lambs were fed the treatment diets for 3 wks following a
7 d adjustment period during which all the animals were fed
the basal diet. After the first supplementation period,
animals were not supplemented with any additional Zn for 4 wks
and then supplementation was resumed for the last wk .

36
Animals weights were obtained at the beginning and at the
end of the experiment . Blood samples were obtained via
jugular venipuncture on d 0 before diet administration and on
d 14, 21, 28, 49 and 55. To obtain serum, samples were
centrifuged at 700 x g for 25 min, supernatant decanted and
frozen until analyzed for Zn and Cu levels. At the end of the
experiment on d 55, animals were stunned with a captive bolt
shot and euthanized by exsanguination .

Liver, pancreas, kidney, bone and bone marrow
(metacarpus), skin, hoof, leg muscle (flexor carpi ulnaris),
and eye were excised and frozen for further mineral analyses.
Zinc and Cu in tissues, blood constituents and diet were

TABLE 4-1. Composition of basal diet
offered to sheep (as fed)a

Ingredient Percentage

Ground yellow corn 59

Cotton seed hulls 21

Soybean meal (44% CP) 12

Alfalfa meal (14% CP) 3

Corn oil 3

Trace mineral saltb 1

Ground limestone 1

Vitamins A and D3C .0008

a Zn analysis biweekly indicated means of 415, 444,
446 and 421 mg/kg for ZnO, ZnS04, ZnLys and ZnMet,
respectively .

b Provided .24 mg of I (KI), .28 mg of Co (CoC03), 82
mg of Fe (FeS04), 38 mg of Cu (CuCl), 48 mg of Mn
(MnS04) and no Zn per kg of diet.
0 Provided 5,000 IU of vitamin A and 500 IU of
vitamin D3 per kg of diet .

37
determined by air acetylene flame atomic absorption
spectrophotometry on a Perkin-Elmer Model 5000 with AS-50.
Metallothionein was measured in liver, pancreas and kidney by
procedures previously described (chapter 3) .

All data were analyzed using SAS (SAS, 1988) . Repeated
measures ANOVA was performed using the general linear model

(GLM) procedure on changes (increase or decrease from d 1) in
serum Zn and Cu concentrations. Tissue Zn and Cu data were
analyzed using GLM procedure. In case of significance (P <

.05) of either serum or tissue mineral concentrations, Waller-
Duncan's K-ratio T test was used for multiple comparisons

(Waller and Duncan, 1969) .

Results

Three of the lambs (2 from the control group and 1 from
the sulfate group) died from unrelated causes. Weight gains
were not different (P > .05) among treatments. Feed intakes
were similar for all groups with no anorexia noted.

When compared to d 1, d 49 serum Zn concentrations had
increased by .20 ± .13 (ig/ml (mean ± SEM) for control sheep
which was lower (P < .05) than those of ZnLys, ZnS04, and ZnO
treatments which had increased by .74 ± .12, .62 ± .07, and
.83 ± .12 |ig/ml, respectively, but was not lower than ZnMet
which increased by .52 ± .16 fig /ml (means shown on Figure 4-
1) . Treatment differences were also seen on d 55 with a
higher (P < .05) serum Zn increase for ZnLys (1.58 ± .28

38
(ig/ml) than ZnMet , ZnO or control treatments (.78 ± .27, .62
± . 1, and .75 ± .26 (ig/ml, respectively), but not ZnS04 (.87
± .17 |ig/ml) . During the depletion phase (d 21 to 49) mean
serum Zn levels did not differ (P > .05) from those levels
before the beginning of depletion (d 21) . Overall serum Cu
levels fell slightly with all treatments (Figure 4-2) . Most
serum concentrations were, however, above the minimum critical
level of .65 (ig/ml, suggested by McDowell et al . (1984) .
There were no treatment effects (P > .05) for any of the
sampling days.

The ZnLys treatment had the highest (P < .05) Zn
accumulation (581, 389, and 340 mg/kg) for kidney, liver and
pancreas, respectively (Table 4-2). Both ZnS04 and ZnMet
treatments had higher (P < .05) liver Zn concentrations (195
and 198 mg/kg, respectively) than the control treatment (127
mg/kg) . Liver Zn concentrations for ZnO were not different (P
> .05) than control, ZnS04 or ZnMet. Kidney Zn concentrations
of both ZnS04 and ZnMet treatments tended (P < .15) to be
higher than controls. The remaining Zn levels for bone, bone
marrow, cornea, skin, hoof and muscle were not different (P >
.05) among treatments. Most of the Zn concentrations for
those tissues were relatively constant among treatments.

Response to treatments in terms of tissue MT (Table 4-3)
were very similar to that of tissue Zn levels. The ZnLys
treatment had the highest (P < .05) MT levels of 79, 167, and
68 (ig MT/g for liver, kidney, and pancreas, respectively.

39

0 5 10 15 20 25 30 35 40 45 50 55
Days on trial

FIGURE 4-1. Mean serum Zn levels for sheep supplemented with
different sources of Zn . SEM (ug/ml) for d 0 .40, d 14 .40,
d 21 .43, d 28 .59, d 49 .34, d 55 .63.

0 5 10 15 20 25 30 35 40 45 50 55
Days on trial

FIGURE 4-2. Mean serum Cu levels for sheep supplemented with
different sources of Zn . SEM (|ig/ml) for d 0 .15, d 14 .16,
d 21 .18, d 28 .15, d 49 .20, d 55 .23.

40

TABLE 4-2 . Mean Zn concentrations in tissues of sheep
supplemented with four sources of Zn (mg/kg, DMBa)

Tissue

Controlb

ZnOc

ZnS04d

ZnMetc

ZnLysc

SEMe

Bone

83

98

97

96

91

13

Bone Marrow

63

79

79

84

73

21

Cornea

7

6

6

7

8

2

Skin

24

27

26

26

31

9

Hoof

94

112

113

105

89

59

Kidney

117f

137f

2 3 4f

226f

581g

131

Liver

127f

140f*g

195g

198g

389h

66

Muscle

260

257

261

259

267

22

Pancreas

86f

107f

1391

135f

340g

110

a DMB= Dry matter basis, bone also fat free, cornea and skin on wet

basis .

b n=6. c n=8. d n=7 .

e SEM = Standard Error of the Mean.

f.g.h Means with different subscripts across row differ (P < .05) .

TABLE 4-3 . Mean metallothionein content of tissues of
sheep supplemented with four sources of Zn (|ig MT/ga)

Tissue

Control

ZnOc

ZnS04d

ZnMetc

ZnLys0

SEMe

Liver

2.0f

4.5£

llf

8.2f

79g

21

Kidney

4.8f

14f

45f

45f

167g

50

Pancreas

1.7*

2.6f

7.9f

llf

68g

36

" |ig MT/g= |ig of Metallothionein per gram of wet tissue.

b n=6. c n=8. d n=7 .

e SEM = Standard Error of the Mean.

t,q Means with different subscripts across row differ (P < .05)

Likewise, both ZnS04 and ZnMet treatments resulted in higher
concentrations than both the control and ZnO groups in the
three tissues but differences were not significant (P > .05) .

41

TABLE 4-4. Mean Cu levels in tissues of sheep
supplemented with four sources of Zn (mg/kg, DMBa)

Tissue

Control13

ZnOc

ZnS04d

ZnMetc

ZnLysc

SEMb

Bone

1.0

.9

.9

1.2

1.1

.3

Bone Marrow

9

10

11

10

6

7

Hoof

3.2

3.3

3.5

3.5

2.4

1.9

Kidney

41

41

66

43

53

31

Liver

1076

1299

1365

1239

1201

381

Muscle

10£

7g

6g

5g

6g

2.3

Pancreas

13

9

10

5

9

4

a DMB= Dry matter basis, bone also fat free basis.

b n=6. c n=8. d n=7 .

e SEM = Standard Error of the Mean.

f,g Means with different subscripts across row differ (P < .05)

Most tissue Cu concentrations did not differ (P > .05)
among treatments and remained relatively constant. Mean
muscle Cu concentration (10 mg/kg), however, was highest (P <
.05) for the control group. There was a large Cu accumulation
in the livers of these animals and was probably due to the
high dietary Cu (70 mg/kg) levels.

Discussion

Unlike in chapter 3, where Zn intake was a minimum of 75
mg/d, Zn levels for the basal diet (16 to 20 mg/kg) for this
trial with sheep were well below the marginal level of 30
mg/kg (NRC, 1985) . Furthermore, since the animals were only
given 1 kg of feed, their actual Zn intake from the basal diet
was only 16 to 20 mg/d.

42
Serum Zn concentrations increased with supplementation,
but these levels did not decrease during the month of
depletion. Therefore, it is impossible to speculate from
these data about tissue Zn retention by the different
treatment groups. Increased retention of Zn in lambs fed
ZnMet had been shown to be greater than that from ZnO
treatment (Spears, 1989) . Mean serum Zn levels were different
on d 49 and 55. Furthermore, on d 55 Zn levels for the ZnLys
treatment had increased more from d 1 than those of all
treatments except ZnS04 . This may imply a higher
bioavailability for these two sources.

Serum Cu concentrations dropped slightly during the
trial. This was unexpected since the basal diet contained
around 70 mg/kg Cu to prevent the adverse effects of the high
levels of Zn supplementation. Since serum Cu content was not
lowered by the high Zn content of the Zn supplemented
treatments, this suggests different absorption routes for
these elements. Furthermore, decreased serum Cu levels were
unexpected because of the accumulation of Cu in the liver of
all the animals. Therefore, the low serum Cu may have
resulted from low hepatic Cu mobilization. There were,
however, no signs of Cu toxicity in any of the animals
studied.

The data suggest that ZnLys has greater bioavailability
as a source of supplemental Zn . This suggestion is made on
the basis of greater accumulation of Zn in the liver, kidney

43
and pancreas of the ZnLys treated animals. The other organic
source (ZnMet) was not different than ZnS04 . There is a
possibility that if the ZnLys treatment had been omitted there
would be differences between the ZnMet and ZnS04 groups
compared to the ZnO and control groups, due to the high values
of the ZnLys group particularly for liver and kidney Zn and MT
levels, which might imply different variances for the
different means. No differences were observed in the bone Zn
deposition for the various treatments. This is unlike the
work of Wedekind and Baker (1990) which showed increased bone
Zn deposition in chicks fed ZnMet relative to ZnO and ZnS04 .
Wedekind et al . (1992) also suggested that bone Zn levels
increased when ZnMet was used compared to ZnS04 or ZnO in
chicks, however, they did not use fat-free bone in their
study. Results reported herein (no differences) also agree
with those of Medeiros et al . (1989) who found that ZnMet did
not influence muscle (longissimus or biceps femoris) Zn
content .

Metallothionein determination was valuable in assessing
the differences among Zn sources. Mean liver, kidney and
pancreas MT levels from the ZnLys treatment ranged anywhere
from about 3 to 40 times greater than the other treatments.
The closest values were those for ZnMet and ZnS04 and the
lowest values were usually those of the negative control group
which was expected. These MT determinations confirm the poor
biological value of ZnO, with tissue concentrations associated

44
with this treatment being very close to the control treatment.
These results indicate the lack of stimulus by the relatively
high Cu levels in the control and ZnO groups on MT levels.
The inability of Cu to act as a stimulus for MT induction is
well documented (Saylor et al., 1980; Peterson and Mercer,
1988) . Since MT present in sheep is not stimulated by Cu,
this may be one of the causes for their high susceptibility to
Cu toxicosis. Furthermore, the limited capacity of sheep to
synthesize MT in the intestinal mucosa (Saylor et al., 1980)
may also be a factor. This limited ability to block Cu
absorption at the intestinal level is supported by the high
levels of Cu in sheep livers in all treatments.

Tissue Cu levels were not greatly affected by Zn
supplementation. There was a decrease from 30 to 50% in
muscle Cu concentrations for animals Zn supplemented compared
to controls, however. Copper muscle concentrations of
controls were probably due to one of two factors or both, the
high dietary Cu levels, or the low dietary Zn levels of the
control diet. A diet of 100 mg/kg Zn has been shown to
decrease liver Cu storage (Pope, 1971) . This decrease did not
occur in this experiment and actually the mean Cu content for
the control group was slightly lower (but not different) than
that of any other group.

Metallothionein has been shown to bind Cu with a very-
high affinity. Therefore, Cu present in liver, kidney and
pancreas of the ZnLys treated animals is stored more in a

45
complex with MT than the Cu present in any of the other
treatments. In a similar manner, because of its relatively
higher MT values, the liver, kidney and pancreas Cu in the
ZnMet and ZnS04 treatments is bound to MT in comparison with
ZnO and the negative control. Despite the higher MT levels in
these tissues, this protein accounts for only a small portion
of the Cu and Zn concentration in these tissues. The
unexplainable factor is that the serum Cu levels were similar
for all treatments and controls should have had higher serum
Cu concentrations. This is expected since serum Cu
(ceruloplasmin) is a hormonally regulated process. The only
consolation is that levels for the control treatment were
increasing (but not different) by the end of the experiment.
Very few studies have evaluated the actual biological
value of different organic Zn sources. Results of this study
indicate that the organic Zn sources can be more (ZnLys) or
equally (ZnMet) available as the best inorganic Zn source.
There were no differences in the particular target pools (or
tissues other than liver, kidney and pancreas) for the Zn
sources, suggesting that Zn from the different sources may be
metabolized equally in those tissues. Zinc from the ZnLys
supplementation may be metabolized differently than that of
the other sources because of its higher levels in the liver,
kidney and pancreas .

46
Implications

Organic sources of Zn (ZnLys and ZnMet) have equal or
greater availability than the most available inorganic source
(ZnS04) and may be metabolized differently in some tissues.
It is suggested that the highly increased synthesis of MT is
proof that ZnLys is a more bioavailable source of Zn.
Research is needed to determine if Cu toxicity in sheep can be
more effectively suppressed with the use of ZnLys.

Summary and Conclusions

A study was conducted to compare supplemental ZnLys,
ZnMet, ZnS04, and ZnO on Zn, Cu and MT concentrations in
various fluids and tissues of 40 wether lambs. Supplemental
Zn (3 60 mg/kg) was fed for 3 wks , withdrawn for 4 wks and then
resumed for another wk . Mineral (Zn and Cu) concentrations
were determined in serum, liver, pancreas, kidney, bone, bone
marrow, hoof, and leg muscle, and only Zn was determined in
skin and cornea. Metallothionein content was determined in
liver, pancreas and kidney. By d 49 serum Zn had increased
less (P < .05) for controls than all but ZnMet, and on d 55 it
had increased more (P < .05) for ZnLys than all but ZnS04 .
There were no treatment effects in serum Cu content, but
overall Cu content fell slightly. The ZnLys treatment had the
highest (P < .05) Zn accumulation (581, 389, and 340 mg/kg)
for kidney, liver and pancreas, respectively. Both ZnS04 and
ZnMet treatments had higher (P < .05) liver Zn concentrations

47
(195 and 198 mg/kg, respectively) than the control treatment
(127 mg/kg) . Mean Zn content of bone, bone marrow, cornea,
skin, hoof and muscle was not different (P > .05) among
treatments. The ZnLys treatment had the highest (P < .05) MT
levels of 79, 167, and 68 (ig MT/g for liver, kidney, and
pancreas, respectively. Mean muscle Cu concentration was
highest (P < .05) for controls (10 mg/kg) . Organic sources of
Zn have equal or greater availability than the most available
inorganic source and may be metabolized differently in some
tissues .

CHAPTER 5

DEVELOPMENT OF ACUTE COPPER POISONING IN

SHEEP FED ORGANIC OR INORGANIC COPPER

Introduction

It is well known that sheep are one of the most sensitive
animals to Cu toxicosis. The exact biochemical etiology of
the Cu toxicity is not well known. Saylor et al . (1980)
suggested that because of the low capability for intestinal
metallothionein (MT) synthesis by sheep, the Cu absorption
process was not as well regulated as that of other species.
Furthermore, it is well known that MT in the tissues of sheep
do not respond to increased inorganic Cu levels (Saylor et
al., 1980; Peterson and Mercer, 1988).

The toxicity, however, can be either chronic or acute
depending on the dosage and time of exposure to the mineral.
Sheep that are supplemented with relatively high doses of
inorganic Cu during an extended period of time may die of
hemolytic crisis. During the first phase of the increased
dose (> 26 mg/d) , the liver and other tissues of the sheep
accumulate Cu . This phase may last from 6 to 10 weeks or
longer. After the tissues are saturated with Cu, the blood
levels of Cu begin to rise, the animals loose their appetite,
develop an excessive thirst and become jaundiced. During this

48

49
hemolytic crisis, the liver becomes cirrhotic and the kidneys
turn very dark, hemoglobin-stained. This crisis eventually
leads to the death of the animal within a few days.

It has been reported that Cu from some organic sources
may be more bioavailable than that from inorganic sources. It
has also been postulated, however, that the supplementation of
a complexed source of copper may retard or even prevent the
onset of this toxicosis. Ishmael et al . (1977) proved that
the supplementation of copper methionate in subcutaneous
injections was not as toxic as Cu Ca EDTA. It was also
hypothesized (Ashmead and Jeppsen, 1993) that the toxicity of
complexed minerals is lower than salts due to stearically
shielding the metals with the amino acids which have bent
around the metals as a consequence of forming the bond.
Spears et al . (1991) hypothesized that certain trace mineral
chelates or complexes may enter different pools in the body
than the inorganic forms. This fact alone may make the Cu
from Cu lysine (CuLys) more available but not as toxic as
CuS04.

Because of the known information on Cu toxicosis, the
tissues to analyze for Cu would include liver and kidney.
Serum Cu and Zn and tissue Zn analyses would also be necessary
because of the known interactions between these minerals.

The objective of this study was to compare toxicity of
CuLys and CuS04 when supplemented at concentrations that would
cause a chronic toxicity to sheep.

50
Materials and Methods

Three crossbred wethers and one crossbred ewe averaging
46 kg were randomly assigned to one of two groups. The two
groups consisted of basal diet + 250 mg/kg of supplemental Cu
from either CuS04 or CuLys . Lambs were housed in individual
wooden pens (1.4 nr) with expanded metal floors in an open
side barn. Feed intake was restricted to 1200 g/hd daily (as-
fed basis) and tap water was available ad libitum. The diet
(Table 5-1) was formulated to be adequate in protein, energy,
vitamins, and minerals for this class of sheep (NRC, 1985) .
Lambs were fed the treatment diets for 4-11 wks

TABLE 5-1. Composition of basal diet
offered to sheep (as fed)a.

Ingredient Percentage

Ground yellow corn 59

Cotton seed hulls 21

Soybean meal (44% CP) 12

Alfalfa meal (14% CP) 3

Corn oil 3

Trace mineral saltb 1

Ground limestone 1

Vitamins A and D3C .0008

a Analysis indicated 290 and 303 mg Cu/kg of feed for
CuS04 and CuLys treatments, respectively. Diets
formulated to provide 250 mg supplemental Cu/kg diet.
b Provided 2 . 4 mg of I (KI ) , .48 mg of Co (CoC03), 82
mg of Fe (FeS04), 48.19 mg of Mn (MnSOJ, 40 mg Zn
(ZnSOJ and no Cu per kg of diet.
c Provided 5,000 IU of vitamin A and 500 IU of
vitamin D, per kg of diet.

51
following a 7 d adjustment period during which all the animals
were fed the basal diet. Protocol for animal care had been
approved by the University of Florida's Institutional Animal
Care and Use Committee.

Blood samples were obtained via jugular venipuncture on
d 1 before animals were administered the concentrate and
biweekly thereafter. To obtain serum, samples were
centrifuged at 700 x g for 25 min, supernatant decanted and
frozen until analyzed for Zn and Cu concentrations. To obtain
hematocrit (HCT) percentages, blood was centrifuged at 700 x
g for 10 min. Serum and blood samples were then submitted to
a lab on the day of withdrawal for creatine kinase (CK) , x-
glutamyl-transferase (GGT) , aspartate amino transferase (AST)
and heinz bodies determinations. At the end of the experiment
on d 78, all surviving animals were stunned with a captive
bolt shot and euthanized by exsanguination .

Parts of the liver and kidney were excised and taken to
the veterinary hospital for necropsy and other parts were
frozen for further mineral analyses . Copper and Zn in
tissues, serum and diet samples were analyzed by air acetylene
flame atomic absorption spectrophotometry on a Perkin-Elmer
Model 5000 with AS-50.

Results

Animal 77 (CuSOJ died of natural causes (related to Cu
toxicosis) on d 28 and was consuming an average of only 20 g/d

52
for 14 d prior to death. Animal 46 (CuLys) was slaughtered on
d 43 after it had only been eating 25 g/d for 5 d. Animal 1
(CuLys) was slaughtered on d 78 after it had only been eating
3 0 g/d for 7 d. The ewe lamb (288, CuSOJ never showed
anorexia or any other sign which reflected a Cu toxicity and
was slaughtered with the last animal to end the experiment.

Overall, serum Cu concentrations rose sharply in all
animals, except 288 (CuS04), shortly before death (Figure 5-
1) . For two of the animals (1, CuLys and 77, CuS04) there was
a sharp decrease in serum Cu immediately before death. There
were no plateaus in the serum Cu response curve of animal 77
which might indicate a decreased ability to control serum Cu
concentration when compared to animals 1 and 46 (CuLys) .

Overall, serum Zn (Figure 5-2) concentrations fluctuated
greatly for all animals but were mostly above the .65 (ig/ml
suggested by McDowell et al . (1984) where a deficiency might
be expected. It is also interesting that for the two CuLys
treated sheep, serum Zn concentrations fell dramatically
before death. The other animal (77) that exhibited signs of
Cu toxicity showed low concentrations but also a rise right
before death.

In general, blood HCT (Figure 5-3) started rising before
death for three of the animals (1, 77 and 288) . For the
animal that died of natural causes, the blood HCT fell
dramatically before death.

53

FIGURE 5-1. Serum Cu concentrations for sheep supplemented
with toxic levels of CuS04 (77 and 288) and CuLys (1 and 46) .

10 20 30 40 50 60

Days on trial

70 80

FIGURE 5-2. Serum Zn concentrations for sheep supplemented
with toxic levels of CuS04 (77 and 288) and CuLys (1 and 46) .

54

FIGURE 5-3. Blood hematocrit for sheep supplemented with
toxic levels of CuS04 (77 and 288) and CuLys (1 and 46) .

1,200
1,000

800

600

400

200

0

;

*- Anim #1 + Anim #46 3K Anim #77 O Anim. #288

....i.. ^ts£tK. . \ .... \ .... \ .... \ ,,,, \ .,, .

0 10 20 30 40 50 60 70 80

Days on trial

FIGURE 5-4. Serum creatine kinase concentrations for sheep
supplemented with toxic levels of CuS04 (77 and 288) and CuLys
(1 and 46) .

55

Overall, serum CK (Figure 5-4) activity decreased with
the beginning of Cu supplementation. Creatine kinase
concentrations were highly increased for animals 1 and 77
which also showed increased serum Cu and HCT at the time of
death. On the other hand animal 4 6 did not show such an
increase in CK levels. There also a peak for animal 1 on d 34
but it went back to the previous concentration by the next
sampling time.

Overall, GGT concentrations (Figure 5-5) rose in the
beginning of the trial but fluctuated throughout for all the
wether lambs. Concentrations of GGT rose for the two wethers
receiving CuLys on d 27 and remained high until death. The
other wether (77) had highly variable GGT level which was low
at the time of death. The GGT level for the ewe was
relatively constant throughout the experiment .

Overall, AST concentrations rose from the beginning of
the experiment for all the wethers (Figure 5-6) . These
concentrations also decreased dramatically before death for
two of the animals (1 and 77) and they remained high for the
other (46) .

Heinz bodies determination was positive only for animal
77 on d 27 with 41% noted. Another interesting detail was
noticed in the blood serum. On d 20 and 23, blood serum for
animal 77 had a mustard color to it and on d 27 it turned to
red which was indicative of the full blown hemolytic crisis.
The animal was found dead by d 30. Furthermore, on d 75,

56

500

400

300

200

100

10 20 30 40 50 60 70 80

Days on trial

FIGURE 5-5. Serum x-glutamyltransf erase levels for sheep
supplemented with toxic levels of CuS04 (77 and 288) and CuLys
(1 and 46) .

2,500

2,000

1,500

1,000

500

30 40 50

Days on trial

80

FIGURE 5-6. Serum aspartate amino transferase levels for
sheep supplemented with toxic levels of CuS04 (77 and 288) and
CuLys (1 and 46) .

57
blood serum for animal 1 also exhibited this mustard color
which turned to red on d 78. In contrast, animal 46 did not
show any abnormal color changes .

Animal 1 had a very high liver Cu concentration (Table 5-
1) which might have been expected since that animal had
received the high Cu diet for 78 d. All other animals (46, 77
and 288) had relatively similar liver Cu levels. Kidney Cu
levels varied with animal 1 having the highest (846 mg/kg) and
animal 288 having the lowest (32 mg/kg) .

Liver Zn concentrations (Table 5-2) were higher for
animals 1 (CuLys) and 77 (CuS04) , the animals suspected of
developing a full-blown Cu toxicosis. Kidney Zn
concentrations were very similar for all animals.

The necropsy report for animal 1 (CuLys), slaughtered on
d 78, indicated a mild chronic multifocal bronchopneumonia
with aspirated foreign material. Also, severe chronic
cholangiohepatitis , severe chronic periportal

TABLE 5-1. Liver and kidney Cu concentrations of
sheep exposed to high Cu levels3.

Animal # Liver (mg/kg) Kidney (mg/kgi

1 (CuLys) 2552 846

46 (CuLys) 985 530

77 (CuS04) 1036 426

288 (CuS04) 937 32

Dry matter basis

58

TABLE 5-2. Liver and kidney Zn concentrations of
sheep exposed to high Cu levels3.

Animal # Liver (mg/kg) Kidney (mg/kg)

1 (CuLys) 191 117

46 (CuLys) 90 123

77 (CuS04) 168 126

288 (CuSOJ 107 122

a Dry matter basis.

and centrilobular hepatocyte necrosis and histiocytic
hyperplasia with megalocytosis , biliary hyperplasia and portal
fibrosis were observed in the liver. In the kidney a mild
chronic lymphocytic pyelonephritis with scattered tubular
casts was reported. The absence of hemoglobinuric nephrosis
suggested that the Cu accumulation in the liver was
subclinical at the time of death.

The necropsy report for the liver of animal 46 (CuLys),
slaughtered on d 41, showed an overall reduction of
hepatocytes and increased fibrous stroma. There was also
severe autolysis and biliary stasis. The report for the liver
of animal 77 (CuS04) , which died on d 30, showed a severe,
diffuse hepatopathy with cytoplasmic Cu accumulation and
individual hepatocyte necrosis. Also there was a severe,
intrahepatic cholestasis and a mild, multifocal, suppurative,
acute hepatitis. The pathological report for animal 288
(CuS04) , slaughtered on d 78, indicated severe chronic
multifocal dystrophic mineralization, fibrosis, and

59
granulomatous inflammation which was most likely due to
aspirated material or metabolic injury to lung collagen. The
liver showed a mild Kupffer cell hyperplasia.

Discussion

Serum Cu concentrations provide a good indicator of Cu
status of ruminants. The liver and kidneys of animal 77 could
not tolerate the dietary Cu as well as the others as indicated
by the relatively low levels of Cu the liver and kidney and
the high serum Cu at the time of death compared to animal 1.

Animal 46 may not have died solely from a Cu toxicosis.
The factors which seem to substantiate this theory include the
low CK levels, the high GGT and AST levels (no decline) , the
failure of HCT to rise, and the absence of hepatitis at the
time of death compared to the animals which exhibited signs of
Cu toxicosis.

Animal 288 was apparently never affected by Cu excess.
The exact cause of this is not known because the animal had
relatively similar liver Cu levels as the other animals but
showed no change in any of the serum enzymes.

Blood HCT percentages show a slight increase in
erythrocytes during the days prior to death which might
reflect a decreased capacity to carry oxygen by the cells. A
dramatic decrease in HCT was seen in animal 77 which
represents the full onset of the hemolytic crisis. This
decline was not seen with the other animals perhaps because

60
they were euthanized after anorexia had been seen for several
days .

The CK analysis reflects myodegradation (Meyer et al . ,
1992) in animals 1 and 77 prior to death which might have been
caused by the excess Cu in the blood being picked up by the
muscle and rupturing the cells. The release of GGT from the
hepatic cells of animal 77 was greater than that of all other
animals. The increased GGT activity represents a cholestasis
(Meyer et al . , 1992) by d 13 for all Cu affected animals. It
was interesting to see animals 46 and 1 survive longer with
such high levels of serum GGT as compared to 77 . The source
of the high AST levels is the liver (Duncan and Prasse, 1977) .
A rapid increase occurred in animal 77 and a slower one for
animals 1 and 46 which is proportional to the number of
hepatocytes damaged. Animal 1 seems able to recover from the
original cholestasis due to decreased AST levels.

The heinz bodies seen in animal 77 before death
represents an increased hemoglobin turnover (Meyer et al . ,
1992) resulting from the hemolytic crisis. The yellow colored
serum appearing before death may be an accumulation of bile
acids and bilirubin (also due to hemolytic crisis) in
peripheral circulation but these tests were not conducted. It
is necessary to conduct another trial to determine MT levels
of animals supplemented with CuLys to evaluate the relatively
increased survivability of those animals.

61
Results of this study do not indicate CuLys to be more or
less toxic than CuS04 . These data do indicate that there were
some plateaus in the different serum enzyme and Cu
concentrations for those animals receiving CuLys, which were
not present with animal 77.

Implications

The use of high Cu supplementation in sheep, from this
small scale study, and the development of toxicity is not
dependant on the source used. The supplementation of CuLys as
a source of "safe" Cu when supplemented in excess is
inconclusive and needs to be further researched.

Summary and Conclusions

A study was conducted to compare toxicity of CuLys and
CuS04 when supplemented at concentrations that would cause a
chronic toxicity to sheep. Four animals, 3 crossbred wethers
and 1 crossbred ewe averaging 4 6 kg were randomly assigned to
250 mg of supplemental Cu from either CuLys or CuS04 . Lambs
were fed the treatment diets for 4-11 wks following a 7 d
adjustment period. Blood samples were taken on d 1 before
animals were administered the concentrate and biweekly
thereafter. Serum and blood samples were analyzed for CK,
GGT, AST, HCT, and heinz bodies. Sections of liver and kidney
were excised and necropsied, and together with serum and diet
were analyzed for Cu and Zn . The liver and kidneys of animal

62
77 (CuS04) could not tolerate the high dietary Cu and resulted
in a rapid increase in serum Cu compared to others . Animal
288 was apparently never affected by Cu excess.
Myodegradation was present in animals 1 and 77 prior to death
and was probably caused by excess Cu in blood. The use of
high Cu supplementation in sheep and the development of
toxicity was not dependant on source used.

CHAPTER 6

INTERACTION OF DIFFERENT ORGANIC AND

INORGANIC ZINC AND COPPER SOURCES

FED TO RATS

Introduction

For many years it has been known that the amount of Zn
absorbed can be influenced by the amount of dietary Cu (Miller
et al . , 1979) . The absorption process of Zn and Cu is not
completely understood. Perhaps the first site for interaction
of Zn and Cu is the intestinal membrane. Subsequently,
through the binding of these minerals to metallothionein (MT)
an additional interaction could occur (Cousins and Hempe,
1990) . This metalloprotein has a higher affinity for Cu than
for Zn .

A recent study evaluated mineral content of rat tissues
fed different levels of inorganic sources of Zn and Cu (Larsen
and Sandstrom, 1992) . There was a high interaction which
affected not only the intestinal absorption, but also
distribution of previously absorbed elements in tissues.

Several products offering minerals complexed with amino
acid are available for mineral supplementation. In contrast
to our knowledge with inorganic forms of Cu and Zn, it is not

63

64
known if Zn and Cu will interfere with each other if they are
provided in complexed rather than inorganic forms .

The purpose of this study was to compare bioavailability,
interactions and retention of complexed and inorganic sources
of Zn and Cu fed to rats.

Materials and Methods

Sixty-three male Charles Sprague-Dawley (CD) strain rats
(Charles River Breeding Laboratories, Wilmington, MA) weighing
71.5 ± 7.3 g (mean ± SEM) were individually housed in
suspended, stainless steel cages in an environmentally
controlled room with a 12 -h light: dark cycle.

Rats were individually fed a purified diet and deionized
water ad libitum. The purified diet (Research Diets, New
Brunswick, NJ) was based on the AIN-76a formulation and
contained the ingredients specified in Table 6-1. The
purified diet contained .34 and .71 mg/kg of Zn and Cu,
respectively. The diet was formulated to be adequate in
protein, energy, vitamins, and minerals for this class of rats
(NRC, 1978) .

Different Zn (Zn methionine, ZnMet ; Zn lysine, ZnLys ; Zn
sulfate, ZnS04) and Cu (Cu lysine, CuLys ; Cu sulfate, CuS04;
Cu oxide, CuO) sources were added to the basal diet at 3 0
mg/kg of Zn and 6 mg/kg of Cu to create a 3 X 3 factorial
experiment (organic sources from Zinpro Corporation, Edina,
MN; inorganic sources from Southeastern Minerals , Bainbridge,

65

TABLE 6-1. Composition of purified
diet fed to rats (As-fed) .a

Ingredient g/kg

Egg white solids 200

Cornstarch 150

Sucrose 503

Cellulose 50

Corn oil 50

AIN-76 mineral mixb,c 35

AIN-7 6 vitamin mixc 10

Biotin t

Choline bitartrate 2

a Zn and Cu analysis indicated (mean ± SD) of

29 ± 2 mg Zn/kg and 5.3 ± .5 mg Cu/kg of all

supplemented diets .

b Contains no Zn or Cu .

e Contents of the mineral and vitamin mix are

specified in appendix Tables A-l and A-2,

respectively .

d Provided .004 g biotin/kg.

GA) . Seven rats were randomly assigned to each of these
treatments .

Supplemented diets were fed for 4 wks at which point four
randomly selected rats from each treatment were sacrificed
(first phase). The rest of the animals were fed the basal
diet (Table 6-1) for an additional week (second phase) and
then sacrificed. Protocol for animal care had been approved
by the University of Florida's Institutional Animal Care and
Use Committee. All rats were anesthetized by inhaling

Metafane™ (Methoxyf lurane) and bled by cardiac puncture.

66
To obtain heparinized plasma, blood was centrifuged at
700 x g for 25 min, supernatant decanted and frozen until
analyzed for Zn and Cu . Tissues were immediately excised.
The liver and both kidneys were frozen at - 80°C, and the rear
leg muscles (biceps femoris, vastus lateralis, and gluteous,
combined) and bones (femur, tibia, and fibula, combined) were
frozen at - 20°C.

Total MT was measured in kidney and liver by the 109Cd2+-
binding method (Eaton and Toal, 1982). The Zn and Cu
concentrations in plasma, liver, kidney, muscle and bone were
measured by air acetylene flame atomic absorption
spectrophotometry on a Perkin-Elmer Model 5000 with AS-50.

All data was analyzed using SAS (SAS, 1988) . Tissue and
plasma Zn, Cu and MT data were analyzed using GLM procedure
and in case of significance (P < .05) Waller-Duncan's K-ratio
T test was used for multiple comparisons (Waller and Duncan,
1969) .

Results

Weight gains were not different (P > .05) among
treatments or experimental phases, but there was a tendency (P
= .07) for CuLys to have a higher ADG than CuS04 for phase 1.
Diet intakes were similar for all groups for both phases with
no anorexia noted. When not mentioned there were no
interaction effects (P > .05).

67
Phase 1

Plasma Zn concentrations of rats were not affected (P >
.05) by Zn or Cu source (Table 6-2) . Plasma Cu

TABLE 6-2. Mean plasma Zn and Cu concentrations
for rats supplemented with different sources of Zn
and Cu (|lg/ml)a.

Sources Zn Cu

ZnS04 2.5 1.0

ZnMet 2.2 .8

ZnLys 2.6 .9

CuO 2.2 .2b

CuS04 2.5 1.2C

CuLys 2J5 1.3C

a SEM are as follows: Zn = .75, Cu = .27.

b,c Means with different subscripts within column and mineral

differ (P < .05) .

concentrations, on the other hand, were lower (P < .05) for
CuO than CuS04 or CuLys supplemented rats.

There were no main effects (Zn or Cu; P > .05) for the Zn
concentrations for most tissues (Table 6-3) . Mean Zn
concentrations were relatively constant for all tissues across
treatments. Bone Zn concentrations, however, were higher (P
< .05) for CuLys than for CuO supplemented rats.

There was an interaction effect for bone Zn
concentrations. Bone Zn concentrations were higher (P < .05)
for CuLys than CuS04 rats that were supplemented with ZnS04 and
CuLys supplementation resulted in higher (P < .05) bone Zn

68
concentrations than did CuO for rats receiving ZnMet (Figure
6-1A) . There were no bone Zn differences (P > .05) from Cu
source for the ZnLys source. Bone Zn concentrations were

TABLE 6-3. Mean tissue Zn concentrations for rats
supplemented with different sources of Zn and Cu
(mg/kg, DMBa

)a \ b

Sources Bone Kidney Liver Muscle

ZnS04
ZnMet
ZnLys
CuO
CuS04
CuLys

149

84

76

58

150

85

77

57

150

84

76

58

147=

85

75

56

150c"d

85

77

58

153d

83

77

58

a DMB= Dry matter basis, bone also fat free.

b SEM are as follows: bone = 6, kidney = 14, liver = 10,

muscle = 8 .

c,d Means with different subscripts within column and mineral

differ (P < .05) .

higher (P < .05) for ZnLys than ZnS04 rats that received CuS04
supplementation, however, ZnLys had the lowest (P < .05) bone
Zn concentrations when CuLys was the Cu supplementation source
(Figure 6-1B) . There were no differences (P > .05) in Zn
source for Zn tissue levels when CuO was the supplemental Cu
source .

All tissue Cu concentrations were affected (P < .05) by
supplemental Cu source (Table 6-4) . In all tissues where Cu
was measured, CuO was the lowest (P < .05) available source of
Cu . Furthermore, CuS04 supplemented rats had higher (P < .05)
Cu concentrations in muscle than from CuLys supplementation.

69

— ZnSO.+ ZnMet * ZnLys

155

E 150

s^ /\"

145

yS ^~r~-~-^i__^^/

ZnLys

CuSO,

CuLys

FIGURE 6-1 A+B. Mean bone (dry, fat free basis) Zn
concentrations for rats supplemented with different sources.
A) (Left) Different Cu sources when supplementing different
Zn sources. B) (Right) Different Zn sources when
supplementing different Cu sources. SEM (mg/kg) 6.0.

TABLE 6-4. Mean tissue Cu concentrations
for rats supplemented with different
sources of Zn and Cu (mg/kg, DMBa)b.

Sources

Kidney Liver Muscle

ZnS04
ZnMet
ZnLys

27

11

7

29

12

6

30

12

6

22c

9C

4C

32d

13d

8d

32d

14d

6e

CuO

CuS04
CuLys

a DMB= Dry matter basis.

b SEM are as follows: kidney = 6, liver = 2, muscle

= 2.

c,a,e Means with different subscripts within column

and mineral differ (P < .05) .

70
Different Zn sources did not affect (P > .05) tissue Cu
contents .

Kidney MT content followed the same pattern as Cu
concentrations with CuO being the lowest (P < .05) MT inducer
(Table 6-5) . There was no effect (P > .05) of Zn source on
tissue MT contents for any tissue and no Cu effect (P > .05)
for liver MT.

TABLE 6-5. Mean tissue metallothionein

concentrations for rats supplemented with

different sources of Zn and Cu ( jLlg
MT/ga)h.

Sources Kidney Liver

ZnS04 77 40

ZnMet 7 6 42

ZnLys 80 3 8

CuO 68c 46

CuS04 82d 38

CuLys 84d 37

a (lg MT/g= |ag of Metallothionein per gram of wet
tissue .

b SEM are as follows: kidney = 10, liver = 12.
c,d Means with different subscripts within column
and mineral differ (P < .05) .

Phase 2

Plasma Zn concentrations of depleted rats were not
affected (P > .05) by Zn or Cu source (Table 6-6) . Plasma Cu
concentrations of depleted rats, on the other hand, were lower
(P < .05) for CuO than CuLys supplemented rats.

71
There were no main effects (Zn or Cu; P > .05) for the Zn
concentrations for most tissues of depleted rats (Table 6-7) .

TABLE 6-6. Mean plasma Zn and Cu concentrations
for rats supplemented with different sources of Zn
and Cu for 4 wks and then depleted for 1 wk
(lig/ml)a.

Sources Zn Cu

ZnS04 1.7 .2

ZnMet 2.0 .4

ZnLys 1.6 .3

CuO 1.8 . lb

CuS04 1.7 .2b'c
CuLys 1^8 .5°

a SEM are as follows: Zn = .34, Cu = .22.

b,c Means with different subscripts within column and mineral

differ (P < .05) .

TABLE 6-7. Mean tissue Zn concentrations for rats
supplemented with different sources of Zn and Cu
for 4 wks and depleted for 1 wk (mg/kg, DMBa)b.

Sources Bone Kidney Liver Muscle

ZnS04

ZnMet

ZnLys

CuO

CuS04

CuLys

153

68

66

53

155

68

69

55

157

62

70

57

153

71c

67

56

158

59d

71

55

154

67cd

67

54

a DMB= Dry matter basis, bone also fat free.

b SEM are as follows: bone = 5, kidney = 9, liver = 5, muscle

= 8.

,r-d Means with different subscripts within column and mineral

differ (P < .05) .

72
Kidney Zn concentrations were lower (P < .05) resulting from
CuS04 supplementation than for CuO supplemented rats.

There was an interaction effect for kidney Zn
concentrations after depletion. Kidney Zn concentrations
after depletion were highest (P < .05) for CuO and lowest (P
< .05) for CuS04 supplementation in the rats also receiving
ZnLys supplementation (Figure 6-2A) . There were no kidney Zn
differences (P > .05) from different Cu source for the ZnMet
or ZnS04 supplemented rats. Kidney Zn concentrations were
higher (P < .05) for ZnLys than ZnS04 supplemented rats that
were also given CuO supplementation, however, ZnLys had the
lowest (P < .05) kidney Zn concentrations when CuS04 was the
Cu source (Figure 6-2B) . There were no differences (P > .05)
in Zn source when CuLys was the Cu source.

Most tissue Cu concentrations after depletion were not
affected (P < .05) by supplemental Cu source (Table 6-8) . In
liver, however, CuO supplemented rats had the lowest (P < .05)
Cu concentration. Different Zn sources did not affect (P >
.05) tissue Cu contents.

There was no effect (P > .05) of Zn or Cu source on
tissue MT contents for any tissue after 1 wk of depletion
(Table 6-9) .

73

ZnLys

CuLys

FIGURE 6-2 A+B. Mean kidney (dry basis) Zn concentrations for
rats supplemented with different sources. A) (Left)
Different Cu sources when supplementing different Zn sources.
B) (Right) Different Zn sources when supplementing different
Cu sources. SEM (mg/kg) 6.0.

TABLE 6-8. Mean tissue Cu concentrations
for rats supplemented with different
sources of Zn and Cu for 4 wks and then
depleted for 1 wk . (mg/kg, DMBa)b.

Sources

Kidney

Liver Muscle

ZnS04
ZnMet
ZnLys

23
26
20

9
10

5
7

CuO

CuS04

CuLys

22
24
23

7c

10d
lld

5
6
7

a DMB= Dry matter basis.

b SEM are as follows: kidney = 8, liver = 2, muscle

= 2.

'"',:i Means with different subscripts within column

mineral differ (P < .05).

74

TABLE 6-9. Mean tissue metallothionein
concentrations for rats supplemented with
different sources of Zn and Cu for 4 wks
and then depleted for 1 wk (fig MT/ga)b.

Sources Kidney Liver

ZnS04 44 30

ZnMet 3 7 32

ZnLys 42 3 0

CuO 43 32

CuS04 4 0 3 3

CuLys 39 27

a (lg MT/g= \lg of Metallothionein per gram of wet
tissue .

b SEM are as follows: kidney = 9, liver = 6, no
differences (P > .05) .

Discussion

Plasma Zn and Cu concentrations for phase 1 of the
experiment were very similar to those noted by Blalock et al .
(1988) when inorganic Zn and Cu supplementation was used at
different levels. Plasma Zn concentrations may have been
slightly high in our study because of limited hemolysis of a
few samples. The lower serum Cu concentration for animals
supplemented with CuO confirms the poor bioavailability of
this source (Kincaid, 1988) . Plasma Zn and Cu concentrations
decreased during the week of depletion. Following a similar
pattern, CuLys supplemented rats had higher plasma Cu
concentration than CuO supplemented rats but this time CuO was
not different than CuS04 which could suggest a higher
retention for CuLys.

75
Since tissue Zn concentrations were not affected by Zn
source, this may suggest equal metabolism of all Zn sources in
those tissues at this level of supplementation. Copper,
however, may be involved in bone Zn deposition since CuO
treated rats, which was less available, had lower bone Zn than
CuLys treated rats. Most tissue Zn concentrations decreased
during depletion, but bone showed no change. Kidney Zn
concentrations of CuO supplemented rats were inexplicably
higher than those of CuS04 . Zinc retention based on tissue
data for the different treatments was not affected.

Mean tissue Cu concentrations reflected the same trends
as plasma concentrations indicating that CuO was less
bioavailable than CuLys or CuS04 . Furthermore, CuS04
supplemented rats had the highest muscle Cu concentrations
which suggests that Cu from this source is taken up by muscle
cells more readily. The only tissue to retain the same
proportions of Cu following depletion to those before the
beginning of depletion was the liver, as liver Cu
concentrations of CuO supplemented rats were lower than other
treatments. Kidney and muscle Cu concentrations, however,
stabilized and there was no difference for the different
sources, suggesting a lower retention for CuLys and CuS04 .
Copper retention in those tissues, represented by the lack of
difference among sources, might also be influenced because Cu
levels were marginal for rat tissues treated with CuO which
had low plasma Cu concentrations.

76
The interaction effects shown in bone following
supplementation indicate increased bone Zn deposition by the
ZnLys and CuLys treatments except when combined, in which case
bone Zn concentrations drop. This observation might support
the theory that when complexed, the mineral is "smuggled"
across the membrane by the other molecule's (in this case
lysine) transport mechanism (Ashmead and Jeppsen, 1993) . This
also seemed to be the case when the sulfate forms were
administered together. Copper has been suggested to have a
role in the mineralization of growing bone, either in a
cuproenzyme with ascorbate oxidase activity, or in its soluble
ionic form (Allen and Solomons, 1984) . It was interesting
that this situation was not noted with other tissues but this
is perhaps due to the fact that regular mineral transport
mechanisms may not have been saturated. Following depletion,
there was also an interaction effect, this time in kidney Zn
concentrations. When ZnLys was the Zn source, CuO treated
rats had the highest kidney Zn concentrations and CuS04 the
lowest, which is puzzling.

Mean MT concentrations were not affected by Zn source
suggesting equal biological values. They were, however,
influenced by Cu source as CuO supplemented rats had lower MT
concentrations. The levels shown in this study following
supplementation were higher and following depletion were
similar to those reported for kidney MT (42 ± 3 fig MT/g) by
Blalock et al . (1988) following a 14 d supplementation period

77
of equal levels of Zn and Cu . Liver levels reported in the
same study (42 ± 3 |ig MT/g) were similar to those of our
supplementation phase but higher than those of depleted rats.
Furthermore, dietary Cu level was reported (Blalock et al . ,
1988) not to affect kidney MT concentration. The present
study, the lowest available Cu source showed the lowest MT
concentration, suggesting that Cu does influence MT expression
when the available dietary Cu is very low. Following the week
of depletion all MT levels stabilized and no differences were
observed for different treatments. These results indicate
that, at adequate supplemental levels, organic sources of Zn
and Cu are metabolized similarly in most aspects as the best
inorganic sources (CuS04 and ZnS04) .

Implications

When supplementing adequate dietary levels of Zn and Cu
(30 and 6 mg/kg, respectively), CuO is less available than
CuLys and CuS04, however under the conditions of this
experiment, organic (ZnMet and ZnLys) and inorganic (ZnSOJ
sources of Zn were similar in bioavailability.

Summary and Conclusions

A study was conducted to compare bioavailability,
interactions and retention of different sources of Zn and Cu
fed to rats. Sixty-three male CD rats were fed individually
a purified diet and deionized water ad libitum. The nine

78
treatments included were all combinations of three Zn (ZnMet,
ZnLys, ZnS04) and three Cu (CuLys, CuS04, CuO) sources added
to the basal diet at 3 0 mg/kg of Zn and 6 mg/kg of Cu forming
a 3 X 3 factorial experiment. After the 4 wk supplementation
phase, 4 randomly selected rats from each treatment were
sacrificed (Phase 1) . The remaining rats were fed the
purified, unsupplemented diet for an additional week (Phase
2 ) and the sacrificed. Mineral (Zn and Cu) concentrations were
determined in plasma, liver, kidney, bone, and muscle and MT
content was determined in liver, and kidney. Plasma Cu
concentrations were lower (P < .05) for CuO than CuS04 and
CuLys supplemented rats. Bone Zn concentrations were higher
(P < .05) for CuLys than for CuO supplemented rats. In all
tissues where Cu was measured, CuO was the lowest (P < .05)
available source of Cu . Furthermore, in muscle, CuS04
supplemented rats had higher (P < .05) Cu concentrations than
CuLys. Kidney MT content followed the same pattern as Cu
concentrations with CuO fed rats having the lowest (P < .05)
MT concentrations. Plasma Cu concentrations of depleted rats
were lower (P < .05) for CuO than CuLys supplemented rats.
Kidney Zn concentrations were lower (P < .05) for CuS04 than
for CuO supplemented rats after depletion. In liver, CuO
supplemented rats had the lowest (P < .05) Cu concentration.
Copper oxide was less available than CuLys and CuS04 when
added in adequate dietary levels, however, organic (ZnMet and
ZnLys) and inorganic (ZnS04) sources of Zn were similar.

CHAPTER 7
GENERAL SUMMARY AND CONCLUSIONS

Four experiments were conducted to compare the biological
availability of different organic and inorganic sources of Zn
and Cu by determining the Zn, Cu and metallothionein (MT)
concentration of various fluids and tissues.

Experiment 1 was a 12 wk experiment conducted to compare
supplemental ZnMet, ZnS04 , and ZnO on Zn, Cu and MT
concentrations in various fluids and tissues of 32 yearling
beef cattle. Supplemental Zn (360 mg/d) was fed for 4 wks,
withdrawn for 4 wks and then resumed for another 4 wks.
Mineral (Zn and Cu) concentrations were determined in serum,
liver, pancreas, kidney, bone, bone marrow, hair, hoof, and
neck muscle and only Zn was determined in erythrocytes, skin
and cornea. Metallothionein levels were determined in liver,
pancreas and kidney. There were no treatment differences (P
> .05) in serum or erythrocyte Zn content. Serum Cu
concentrations tended to decrease with all treatments. There
were no treatment differences (P > .05) in Zn and Cu tissue
concentrations and liver, kidney and pancreas MT
concentrations. Tissue Cu concentrations did not decline in

79

80
the supplemented treatments when compared to controls. It was
concluded that, at adequate levels of dietary Zn,
bioavailability of supplemental Zn source may be of less
importance than when added to low Zn diets or at higher
supplemental levels.

Experiment 2 was conducted to compare supplemental ZnLys,
ZnMet, ZnS04, and ZnO on Zn, Cu and MT concentrations in
various fluids and tissues of 40 wether lambs. Supplemental
Zn (3 60 mg/kg) was fed for 3 wks , withdrawn for 4 wks and then
resumed for another wk . Mineral (Zn and Cu) concentrations
were determined in serum, liver, pancreas, kidney, bone, bone
marrow, hoof, and leg muscle and only Zn was determined in
skin and cornea. Metallothionein content was determined in
liver, pancreas and kidney. By d 49 serum Zn had increased
less (P < .05) for controls than all but ZnMet, and by d 55
had increased more (P < .05) for ZnLys than all but ZnS04 .
There were no treatment effects in serum Cu content, but
overall Cu content fell slightly. The ZnLys treatment
produced the highest (P < .05) Zn accumulation in kidney,
liver and pancreas. Both ZnS04 and ZnMet treatments produced
higher (P < .05) liver Zn concentrations than the control
treatment. Mean Zn content of bone, bone marrow, cornea,
skin, hoof and muscle was not different (P > .05) among
treatments. The ZnLys treatment produced the highest (P <
.05) MT levels for liver, kidney, and pancreas. Mean muscle
Cu concentration was highest (P < .05) for controls (10

81
mg/kg) . For this experiment with sheep, organic sources of Zn
were of equal availability or more available than the most
available inorganic source and may be metabolized differently
in some tissues.

Experiment 3 was conducted to compare toxicity of CuLys
and CuS04 when supplemented at concentrations that would cause
a chronic toxicity to sheep. Three crossbred wethers and 1
crossbred ewe averaging 46 kg were randomly assigned to 250 mg
of supplemental Cu from either CuLys or CuS04 . Lambs were fed
the treatment diets for 4-11 wks following a 7 d adjustment
period. Blood samples were taken on d 1 before treatment
began and biweekly thereafter. Serum and blood samples were
analyzed for CK, GGT, AST, and heinz bodies. Sections of
liver and kidney were excised and autopsied, and together with
serum and diet analyzed for Cu and Zn . The liver and kidneys
of one of the CuS04 treated sheep could not tolerate the high
dietary Cu as did the others, resulting in a rapid elevation
of serum Cu . The other CuS04 treated sheep was apparently
never affected by Cu excess. Myodegradation was present in
one animal from each treatment prior to death probably caused
by excess Cu in blood and muscle. The use of high Cu
supplementation in sheep and the development of toxicity from
this limited number of animals was not dependant on source
used.

Experiment 4 was divided into two phases . Phase 1 was
conducted to compare bioavailability of different sources of

82
Zn and Cu by evaluating Zn, Cu and MT concentrations of
selected tissues and plasma, and if interaction can be reduced
by use of different sources. Phase 2 was conducted to compare
Cu and Zn retention time after termination of supplementation.
Sixty-three male CD rats were randomly assigned to one of nine
treatments which were all combinations of three Zn (ZnMet,
ZnLys, ZnS04) and three Cu (CuLys, CuS04, CuO) sources added
to the basal diet at 30 mg/kg of Zn and 6 mg/kg of Cu, to
create a 3 X 3 factorial experiment. After the 4 wk
supplementation phase, 4 randomly selected rats from each
treatment were sacrificed (Phase 1) . The remaining rats were
fed the purified, unsupplemented diet for an additional week
(Phase 2) . Mineral (Zn and Cu) concentrations were determined
in plasma, liver, kidney, bone, and muscle and MT content was
determined in liver, and kidney. Plasma Cu concentrations
were lower (P < .05) for CuO than CuS04 and CuLys supplemented
rats. Bone Zn concentrations were higher (P < .05) for CuLys
than for CuO supplemented rats. In all tissues where Cu was
measured, CuO was the lowest (P < .05) available source of Cu.
Furthermore, in muscle, CuS04 supplemented rats had higher (P
< .05) Cu concentrations than CuLys. Kidney MT content
followed the same pattern as Cu concentrations with CuO
producing the lowest (P < .05) MT concentrations. Plasma Cu
concentrations of depleted rats were lower (P < .05) for CuO
than CuLys supplemented rats. Kidney Zn concentrations were
lower (P < .05) for CuS04 than for CuO supplemented rats after

83
depletion. In liver, CuO supplemented rats had the lowest (P
< .05) Cu concentration. Copper oxide was less available than
CuLys and CuS04 when added in adequate dietary levels,
however, organic (ZnMet and ZnLys) and inorganic (ZnS04)
sources of Zn were similar.

APPENDIX

TABLE A-l. AIN-76A mineral mix without added Zn or Cu
(As-fed)a.

Ingredient g g/35 g

Calcium Phosphate, Dibasic (29.5% Ca,
22.8% P)

Magnesium Oxide (60.3% Mg)

Potassium Citrate, 1 H20 (36.2% K)

Potassium Sulfate (44.9% K, 18.4% S)

Sodium Chloride (39.3% Na, 60.7% CI)

Chromium Potassium Sulfate, 12 H-,0
(10.4% Cr)

Potassium Iodate (59.3% I)

Ferric Citrate (21.2% Fe)

Manganous Carbonate (47.8% Mn)

Sodium Selenite (45.7% Se)

Sucrose

Total

a Mineral mix used in rat experiment, Chapter 6

500

Ca 5.2

P 4

24

Mg .5

220

K 3.6

52

S .33

74

Na 1

mg/3 5 g

.55

Cr 2

.01

I .2

6

Fe 45

3 .5

Mn 5 9

.01

Se .16

19.93

1000

84

.8

4, 000

1

1,000

10

50

mg / 1 0 g

TABLE A-2 . AIN-76A vitamin mix (As-fed) a.

Ingredient g IU/10 g

Vitamin A Palmitate (500,000 IU/g)
Vitamin D3 (100,000 IU/g)
Vitamin E Acetate (500 IU/g)

Menadione sodium Bisulfite (62.5% .08 .5

Menadione)

Biotin (1.0%)

Folic Acid

Nicotinic Acid

Calcium Pantothenate

Pyridoxine-HCl

Riboflavin

Thiamin HC1

Cyanocobalamin (.1%)

Sucrose

Total

a Vitamin used in rat experiment, Chapter 6

2

.2

.2

2

3

30

1.6

16

.7

7

.6

6

.6

6

ug

/10 g

1

10

978.42

1000

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Bettger, W.J., and B.L. O'Dell. 1981. A critical
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BIOGRAPHICAL SKETCH

Luis Xavier Rojas was born on September 16, 1968, in
Falls Church, Virginia, U. S. A. He then moved to Caracas,
Venezuela. He attended high school at Institutos Educacio-
nales Asociados in Caracas until the 10th grade. He went on
to finish high school at Rents Hill School in Rents Hill,
Maine, in order to learn the English language and graduated in
May, 1985. He entered the University of Tennessee at Martin
in September, 1985, and was awarded the degree of Bachelor of
Science in Agriculture in May, 1989.

In August, 1989, he entered the University of Florida as
a postbaccalaureate student. In January, 1990, he was
accepted to the Graduate School at the University of Florida
and began studies in the area of animal nutrition. He
received the degree of Master of Science in animal science in
August 1992. His thesis work was focused on the mineral
status of Venezuelan cattle. During the same year he was
awarded a teaching assistantship and began studies leading to
the degree of Doctor of Philosophy at the University of
Florida. At present he is a candidate to receive a Ph.D. in
animal science in May 1994. In the future he plans to be a
member of the faculty at La Universidad de Oriente in Maturin,
Venezuela and continue working in the same area of study.

98

99

He has helped to teach part of the animal nutrition

class. He is a member of Gamma Sigma Delta honor society of

agriculture, and has been nominated Sigma Xi honor scientific

society and to Alpha Zeta honor society.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

i.Kfi*0*JK

L. R. McDowell, Chair
Professor of Animal Science

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

H\f Conrad
'Professor of Animal Science

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

^O £> • t^>&>

Doug 1 a k B . Ba t e s
Associate Professor of
Animal Science

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

Cousins
Boston Family Professor of
Human Nutrition

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

Frank G. Martin
Professor of Statistics

This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy. A .

April 1994 ^^ &_

Dean, College of
Agriculture

Dean, Graduate School

LP
WO

UNIVERSITY OF FLORIDA

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