Relative bioavailability of different organic and inorganic zinc and copper sources in ruminants and rats

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Relative bioavailability of different organic and inorganic zinc and copper sources in ruminants and rats
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xii, 99 leaves : ill. ; 29 cm.
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Rojas, Luis X., 1968-
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Ruminants -- Physiology   ( lcsh )
Rats -- Physiology   ( lcsh )
Minerals in animal nutrition   ( lcsh )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1994.
Bibliography:
Includes bibliographical references (leaves 86-97).
Statement of Responsibility:
by Luis X. Rojas.
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Typescript.
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Vita.

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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.














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
Zinc . .
Absorption . .
Transport and Tissue Uptake
Bioavailability of Sources


Copper
Copper . . .
Absorption . .
Transport and Tissue Uptake .
Bioavailability of Sources .
Bioavailability of Other Trace Mineral
Complexes . .

CHAPTER 3
RELATIVE BIOAVAILABILITY OF ZINC METHI'OIIIE AND TWO
INORGANIC ZINC SOURCES FED TO CATTLE . .
Introduction . .. .
Materials and Methods . .
Results . . .
Discussion . . .
Implications . . .
Summary and Conclusions . .


I I I I







CHAPTER 4
RELATIVE BIOAVAILABILITY OF TWO
ZINC SOURCES FED TO SHEEP .
Introduction .
Materials and Methods .
Results . .
Discussion .
Implications .
Summary and Conclusions


ORGANIC AND


TWO


INORGANIC


CHAPTER 5
DEVELOPMENT OF ACUTE COPPER POISONING IN SHEEP FED ORGANIC
OR INORGANIC COPPER . . .
Introduction . . .
Materials and Methods . .
Results . . .
Discussion . . .
Implications . . .
Summary and Conclusions . .


CHAPTER 6
INTERACTION OF DIFFERENT ORGANIC AND
COPPER SOURCES FED TO RATS .
Introduction . .
Materials and Methods .
Results . .
Discussion . .
Implications . .
Summary and Conclusions .


INORGANIC ZINC AND


CHAPTER 7
GENERAL SUMMARY AND CONCLUSIONS .

APPENDIX . . .

REFERENCE LIST . .


. 79

. 84

. 86


BIOGRAPHICAL SKETCH ..













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 .. 30

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

TABLE 6-4. Mean tissue Cu concentrations for rats
supplemented with different sources of Zn and Cu

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

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 .

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 . .

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. .

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 .

TABLE A-1. AIN-76A mineral mix without added Zn or Cu
(As-fed) . . .

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


viii













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 CuSO4 (77 and 288)
and CuLys (1 and 46). .. 53

FIGURE 5-2. Serum Zn concentrations for sheep
supplemented with toxic levels of CuSO4 (77 and 288)
and CuLys (1 and 46). .. 53

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

FIGURE 5-4. Serum creatine kinase concentrations for
sheep supplemented with toxic levels of CuSO4 (77
and 288) and CuLys (1 and 46). . 54

FIGURE 5-5. Serum t-glutamyltransferase levels for
sheep supplemented with toxic levels of CuSO4 (77
and 288) and CuLys (1 and 46). . 56

FIGURE 5-6. Serum aspartate amino transferase levels
for sheep supplemented with toxic levels of CuSO4
(77 and 288) and CuLys (1 and 46). . 56







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













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 completed (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, ZnSO4, and ZnO

(360 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, ZnSO4, 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 ZnSO4

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 CuSO4. One of the CuSO4 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 ZnSO4 (30 mg Zn/kg) and CuLys, CuSO4, 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 CuSO4 and CuLys. Bone Zn was

higher for CuLys than CuO. The CuO T was lowest in BAV and

kidney. Rats supplemented with CuSO4 had higher muscle Cu

than with CuLys. After depletion, plasma Cu was lower for CuO

than CuLys, kidney Zn lower for CuSO4 than for CuO which in

turn had the lowest liver Cu. Amino acid completed 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.


xii













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 completing 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 completed 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, 1920). 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









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 200C, and melting and boiling

points of 419.5 and 9060C, 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.









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 a2-macroglobulin







12

(Parisi and Vallee, 1970), transferring (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 ZnSO4 (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 ZnSO4 (Wedekind and Baker, 1990). In another study

(Wedekind et al., 1992) Zn methionine (ZnMet) was reported to

be better than both ZnSO4 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 ZnSO4 (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 ZnSO4

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 ZnSO4 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 29 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

50 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 I-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 completed 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 CuSO4, CuCO3, CuC12 or Cu(NO3)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 CuO and CuO forms were

less available (Lassiter and Bell, 1960). In the growing

chick, Cu from CuI and CuO were 82 and 76%, respectively, as

available as CuSO4 (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 CuSO4 is more available

than CuO. Baker et al. (1991), however, suggested similar

bioavailability for Cu from a Cu lysine (CuLys) complex to

that of CuSO4 in chicks. Liver Cu has been reported to

increase more rapidly when cattle were supplemented a Cu

proteinate versus CuSO4 (Clark et al., 1993). In studies with

weanling pigs (Coffey et al., 1992; Coffey et al., 1993), it

was suggested that CuLys and CuSO4 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, CuSO4, CuCO3, and

Cu acetate sources were of similar bioavailability. Nockels

et al. (1993) suggest that CuLys is better retained than that

of CuSO4 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 CuSO4 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 CuSO4 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 CuSO4 as to their effect on the Cu status of Cu

depleted steers fed diets high in Mo. Ward et al. (1993)

supplemented CuLys or CuSO4 to growing steers fed a diet with

or without supplemental Mo and S. They found no difference

between CuLys and CuSO4 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 completed 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

MnSO4 but more available than MnO. The relative

bioavailability of Mn proteinate was similar to that of MnSO4

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

MnSO4.

Organic iodine (ethylenediamine dihydroiodide)

supplemented mice had similar macrophage phagocytosis to NaIO3

and NaI supplemented mice (Siddiqui et al., 1993).

In a toxicity and tissue retention study conducted with

rats, Na2SeO4 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 completed 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

completed with amino acids are available for mineral

supplementation. Zinc methionine (ZnMet) can bypass ruminal

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 ZnSO4 (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,








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, ZnSO4, 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

m2, 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), ZnSO4

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) 14.30
Dicalcium phosphate 1.05
Saltb .29
Trace mineral mixc .26
Vitamins A and D3d .03
a Zn analysis biweekly indicated means of 248, 251 and
256 mg/kg for ZnMet, ZnSO4 and ZnO, respectively. Hay
intake ranged from 2 to 5 kg (as-fed).
b Provided 2.1 g of NaCl per kilogram of diet.
c Provided .24 mg of I (KI), .28 mg of Co (CoCO,), 82 mg
of Fe (FeSO4), 38 mg of Cu (CuCl), 48 mg of Mn (MnSO4)
and no Zn per kg of diet.
d Provided 5,000 IU of vitamin A and 500 IU of vitamin
D3 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

metacarpuss), 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 'lgCd2-hemoglobin affinity assay (Eaton and Toal,

1982). For this procedure .2 g of the tissue was homogenized

with 4 volumes of cold 10mM Tris-HCl buffer (pH 7.4), with a

Potter-Elvehjem glass-teflon tissue grinder, and centrifuged

(40,000 x g; 10 min; 40C). The supernatant was then heated

(1000C; 5 min) and centrifuged (10,000 x g; 5 min). Next, 200







26

p1 of Cd solution (2 gg Cd and .5 pCi 09Cd per ml of 10 mM

Tris-HC1 buffer, pH 7.4) were added to a 200 p1 aliquot of the

supernatant and the mixture was incubated (10 min; room

temperature). Finally, 100 1l of 2% hemoglobin were added.

The sample was then heated (1000C; 2 min) and centrifuged

(10,000 x g; 5 min). This step was then repeated and 100 1l

of the supernatant and standard solutions were placed in a

Gamma Spectrometer Model 4000 (Beckman Instruments, Inc.) to

measure 10Cd 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 gg/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






















0.8 .



0.6 ------------------


0.4
0 10 20 30 40 50 60 70 80
Days on trial


FIGURE 3-1. Mean serum Zn for cattle supplemented with
different sources of Zn. SEM (gg/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.


,,2.2


2


1.8


Days on trial


FIGURE 3-2. Mean erythrocyte Zn for cattle supplemented with
different sources of Zn. SEM (gg/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.




























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 (pg/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 i]
supplemented with three sources of Zi

Tissue Control ZnMet

Boned 60 60
Bone Marrowe 143 137
Cornea 2.8 2.3

Skin 1.9 1.5
Hair 86 88
Hoof 77 74
Kidney 72 67
Liver 111 116
Muscled 190 179
Pancreas 65 66
a DMB= Dry matter basis, bone also fat free,
basis.
b No difference among treatments (P > .05)
SMetacarpus.
d Sterno mandibularis.


n tisst
n (mg/k

ZnSO4

61
133
3.2

1.8
86
70
69
118
182
66
cornea


ies of cattle
:g, DMBa)

ZnO SEM

62 8
133 22
2.8 .97
2.2 1.4
84 8
81 10
69 5
114 18
192 25
62 10
and skin on wet












TABLE 3-3. Mean Cu levels in tissues of cattle
supplemented with three sources of Zn (mg/kg, DMBa)
Tissueb Control ZnMet ZnSO4 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 (gg MT/ga)
Tissueb Control ZnMet ZnSO4 ZnO SEM
Liver 140 125 108 139 57
Kidney 68 63 63 56 19
Pancreas 43 46 29 44 18
a gg MT/g= gg 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.









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

ZnSO4. Wedekind et al. (1992) also suggested that bone Zn

levels increased when ZnMet was used compared to ZnSO4 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, ZnSO4, and ZnO on Zn, Cu and MT concentrations in

various fluids and tissues of 32 yearling 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 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







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

360 mg/d of supplemental Zn: ZnMet, Zn lysine (ZnLys; Zinpro

Corporation, Edina, MN), ZnSO4 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 m2) 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

metacarpuss), 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 salt 1
Ground limestone 1
Vitamins A and D3c .0008
Zn analysis biweekly indicated means of 415, 444,
446 and 421 mg/kg for ZnO, ZnSO4, ZnLys and ZnMet,
respectively.
b Provided .24 mg of I (KI), .28 mg of Co (CoCO3), 82
mg of Fe (FeSO4), 38 mg of Cu (CuCl), 48 mg of Mn
(MnSO4) and no Zn per kg of diet.
c 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 |Lg/ml (mean SEM) for control sheep

which was lower (P < .05) than those of ZnLys, ZnSO4, and ZnO

treatments which had increased by .74 .12, .62 .07, and

.83 .12 gg/ml, respectively, but was not lower than ZnMet

which increased by .52 .16 gg/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

gg/ml) than ZnMet, ZnO or control treatments (.78 .27, .62

.1, and .75 .26 gg/ml, respectively), but not ZnSO4 (.87

.17 gg/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 gg/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 ZnSO4 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, ZnSO4 or ZnMet. Kidney Zn concentrations

of both ZnSO4 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 pg MT/g for liver, kidney, and pancreas, respectively.



























0.5'
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 (Rg/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 (gg/ml) for d 0 .15, d 14 .16,
d 21 .18, d 28 .15, d 49 .20, d 55 .23.














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


supplemented with four sources of Zn


(mg/kg, DMBa)


Tissue Controlb ZnOc

Bone 83 98
Bone Marrow 63 79
Cornea 7 6
Skin 24 27
Hoof 94 112
Kidney 117f 137f
Liver 127f 140fg
Muscle 260 257
Pancreas 86f 107f
a DMB= Dry matter basis, bone also fat
basis.
b n=6. c n=8. d n=7.


ZnSO4d
97
79
6
26
113
234f
1959
261
139f
free,


ZnMetc

96
84
7
26
105
226f
1989
259
135f
cornea and


ZnLysc SEMe

91 13
73 21
8 2
31 9
89 59
581g 131
389h 66
267 22
3409 110
skin on wet


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 (pig MT/ga)

Tissue Controlb ZnOc ZnSO4d ZnMetc ZnLysc SEIm

Liver 2.0' 4.5f llf 8.2f 799 21
Kidney 4.8f 14f 45' 45f 167g 50
Pancreas 1.7f 2.6f 7.9f 11i 68' 36
a gg MT/g= gg of Metallothionein per gram of wet tissue.
b n=6. c n=8. d n=7.
e SEM = Standard Error of the Mean.
f'9 Means with different subscripts across row differ (P < .05).



Likewise, both ZnSO4 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).










TABLE 4-4. Mean Cu levels in tissues of sheep
supplemented with four sources of Zn (mg/kg, DMBa)
Tissue control 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 l0f 79 6g 59 69 2.3
Pancreas 13 9 10 5 9 4
a DMB= Dry matter basis, bone also fat free basis.
b n=6. n=8. d n=7.
SSEM = Standard Error of the Mean.
f' 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 ZnSO4. 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 ZnSO4. There is a

possibility that if the ZnLys treatment had been omitted there

would be differences between the ZnMet and ZnSO4 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 ZnSO4.

Wedekind et al. (1992) also suggested that bone Zn levels

increased when ZnMet was used compared to ZnSO4 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 ZnSO4 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 ZnSO4 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.









Implications


Organic sources of Zn (ZnLys and ZnMet) have equal or

greater availability than the most available inorganic source

(ZnSO4) 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 (360 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 ZnSO4.

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 ZnSO4 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 gg 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 completed 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

completed 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

CuSO4.

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 CuSO4 when supplemented at concentrations that would

cause a chronic toxicity to sheep.










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 CuSO4 or CuLys. Lambs were housed in individual

wooden pens (1.4 m2) 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
CuSO4 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 (CoCO3), 82
mg of Fe (FeSO4), 48.19 mg of Mn (MnSO4), 40 mg Zn
(ZnSO4) and no Cu per kg of diet.
c Provided 5,000 IU of vitamin A and 500 IU of
vitamin D3 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), T-

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 (CuSO4) 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

30 g/d for 7 d. The ewe lamb (288, CuSO4) 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 (CuSO4), shortly before death (Figure 5-

1). For two of the animals (1, CuLys and 77, CuSO4) 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 lg/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.




























0 10 20 30 40 50 60 70 80
Days on trial


FIGURE 5-1. Serum Cu concentrations for sheep supplemented
with toxic levels of CuSO4 (77 and 288) and CuLys (1 and 46).


Days on trial


FIGURE 5-2. Serum Zn concentrations for sheep supplemented
with toxic levels of CuSO4 (77 and 288) and CuLys (1 and 46).




























Days on trial


FIGURE 5-3. Blood hematocrit for sheep supplemented with
toxic levels of CuSO4 (77 and 288) and CuLys (1 and 46).


Days on trial


FIGURE 5-4. Serum creatine kinase concentrations for sheep
supplemented with toxic levels of CuSO4 (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 46 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,






















200 ---...


100 ............ .... ....



0 10 20 30 40 50 60 70 80
Days on trial


FIGURE 5-5. Serum T-glutamyltransferase levels for sheep
supplemented with toxic levels of CuSO4 (77 and 288) and CuLys
(1 and 46)


2,500
0Anim. #1 Anim. #46 X Anim. #77 Anim. #288


2,000 --.................... ........


1,500 -...


1,000 .


500 ..



0 10 20 30 40 50 60 70 80
Days on trial


FIGURE 5-6. Serum aspartate amino transferase levels for
sheep supplemented with toxic levels of CuSO4 (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 (CuSO4), 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 levels.
Animal # Liver (mg/kg) Kidney (mg/kg)
1 (CuLys) 2552 846
46 (CuLys) 985 530
77 (CuSO4) 1036 426
288 (CuSO4) 937 32
a Dry matter basis.










TABLE 5-2. Liver and kidney Zn concentrations of
sheep exposed to high Cu levels
Animal # Liver (mg/kg) Kidney (mg/kg)
1 (CuLys) 191 117
46 (CuLys) 90 123
77 (CuS04) 168 126
288 (CuSO4) 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 (CuSO4), 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

(CuSO4), 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 CuSO4. 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

CuSO4 when supplemented at concentrations that would cause a

chronic toxicity to sheep. Four animals, 3 crossbred wethers

and 1 crossbred ewe averaging 46 kg were randomly assigned to

250 mg of supplemental Cu from either CuLys or CuSO4. 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 (CuSO4) 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 completed with amino

acid are available for mineral supplementation. In contrast

to our knowledge with inorganic forms of Cu and Zn, it is not







64

known if Zn and Cu will interfere with each other if they are

provided in completed rather than inorganic forms.

The purpose of this study was to compare bioavailability,

interactions and retention of completed 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, ZnSO4) and Cu (Cu lysine, CuLys; Cu sulfate, CuSO4;

Cu oxide, CuO) sources were 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 (organic sources from Zinpro Corporation, Edina,

MN; inorganic sources from Southeastern Minerals, Bainbridge,










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-76 vitamin mixc 10
Biotin d
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.
SContents of the mineral and vitamin mix are
specified in appendix Tables A-i 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

MetafaneTM (Methoxyflurane) 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 800C, and the rear

leg muscles (biceps femoris, vastus lateralis, and gluteous,

combined) and bones (femur, tibia, and fibula, combined) were

frozen at 200C.

Total MT was measured in kidney and liver by the '09Cd2'-

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 CuSO4 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).










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 (gg/ml)a
Sources Zn Cu


ZnSO4 2.5 1.0
ZnMet 2.2 .8
ZnLys 2.6 .9
CuO 2.2 .2b

CuSO4 2.5 1.2c
CuLys 2.6 1.3c
a SEM are as follows: Zn = .75, Cu = .27.
bc Means with different subscripts within column and mineral
differ (P < .05).


concentrations, on the other hand, were lower (P < .05) for

CuO than CuSO4 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 CuSO4 rats that were supplemented with ZnSO4 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)b
Sources Bone Kidney Liver Muscle
ZnS04 149 84 76 58
ZnMet 150 85 77 57
ZnLys 150 84 76 58

CuO 147c 85 75 56

CuSO4 150cd 85 77 58
CuLys 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.
cd Means with different subscripts within column and mineral
differ (P < .05).



higher (P < .05) for ZnLys than ZnSO4 rats that received CuSO4

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, CuSO4 supplemented rats had higher (P < .05)

Cu concentrations in muscle than from CuLys supplementation.









69




160 160
CuO + CuSO,- CuLys ZnSO-+ ZnMet ZnLys

... ........ .. .. .. 1


S150 .. 6 150


145 .....-- .......- 145 -


140 140
ZnSO4 ZnMet ZnLys CuO CuSO4 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

ZnSO4 27 11 7

ZnMet 29 12 6

ZnLys 30 12 6

CuO 22c 9C 4C

CuSO4 32d 13d 8d

CuLys 32d 14d 6e

SDMB= Dry matter basis.
b SEM are as follows: kidney = 6, liver = 2, muscle
= 2.
c,d,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 (Lg
MT/ga)b
Sources Kidney Liver

ZnSO4 77 40
ZnMet 76 42
ZnLys 80 38

CuO 68c 46

CuSO4 82d 38
CuLys 84d 37
a gg MT/g= gg of Metallothionein per gram of wet
tissue.
b SEM are as follows: kidney = 10, liver = 12.
c', 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.










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
(pg/ml)a

Sources Zn Cu


ZnSO4
ZnMet


1.7
2.0


ZnLys 1.6 .3

CuO 1.8 .Ib


CuSO4
CuLys


1.7
1.8


.2b.c


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


ZnSO4

ZnMet


153

155


ZnLys 157 62 70 57

CuO 153 71C 67 56


CuSO4
CuLys


158
154


59d
67c,d


a DMB= Dry matter basis, bone also fat free.
b SEM are as follows: bone = 5, kidney = 9, liver = 5, muscle
= 8.
c,a Means with different subscripts within column and mineral
differ (P < .05).







72

Kidney Zn concentrations were lower (P < .05) resulting from

CuSO4 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 ZnSO4 supplemented rats. Kidney Zn concentrations were

higher (P < .05) for ZnLys than ZnSO4 supplemented rats that

were also given CuO supplementation, however, ZnLys had the

lowest (P < .05) kidney Zn concentrations when CuSO4 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




s0 CuO +CuSO*CuLys -ZnSO +ZnMet *-ZnLys

71 70 .


560E 560

50 -- 50


40 40
ZnSO ZnMe ZnLys CuO CuSO4 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

ZnSO4 23 8 6

ZnMet 26 9 5

ZnLys 20 10 7

CuO 22 7c 5

CuSO4 24 10d 6

CuLys 23 Ild 7

a DMB= Dry matter basis.
b SEM are as follows: kidney = 8, liver = 2, muscle
=2.
cd Means with different subscripts within column
mineral differ (P < .05).










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 (plg MT/ga)b.
Sources Kidney Liver
ZnSO4 44 30
ZnMet 37 32
ZnLys 42 30
CuO 43 32
CuSO4 40 33
CuLys 39 27
a gg MT/g= gg 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 CuSO4 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 CuSO4. 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 CuSO4. Furthermore, CuSO4

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 CuSO4.

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 completed, 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 CuSO4 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 gg 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 gg 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 (CuSO4 and ZnSO4).


Implications


When supplementing adequate dietary levels of Zn and Cu

(30 and 6 mg/kg, respectively), CuO is less available than

CuLys and CuSO4, however under the conditions of this

experiment, organic (ZnMet and ZnLys) and inorganic (ZnSO4)

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, ZnSO4) and three Cu (CuLys, CuSO4, CuO) sources added

to the basal diet at 30 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 CuSO4 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, CuSO4

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 CuSO4 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 CuSO4 when

added in adequate dietary levels, however, organic (ZnMet and

ZnLys) and inorganic (ZnSO4) 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, ZnSO4, 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







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, ZnSO4, and ZnO on Zn, Cu and MT concentrations in

various fluids and tissues of 40 wether lambs. Supplemental

Zn (360 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 ZnSO4.

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 ZnSO4 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 CuSO4 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 CuSO4. 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 CuSO4 treated sheep could not tolerate the high

dietary Cu as did the others, resulting in a rapid elevation

of serum Cu. The other CuSO4 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, ZnSO4) and three Cu (CuLys, CuSO4, 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 CuSO4 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, CuSO4 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 CuSO4 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 CuSO4 when added in adequate dietary levels,

however, organic (ZnMet and ZnLys) and inorganic (ZnSO4)

sources of Zn were similar.














APPENDIX


TABLE A-1. AIN-76A mineral mix without added Zn
(As-fed)a


Ingredient

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% Cl)


g
500

24
220
52
74


or Cu


g/35 g
Ca 5.2
P 4
Mg .5
K 3.6
S .33
Na 1
mg/35 g


Chromium Potassium Sulfate, 12 H20 .55 Cr 2
(10.4% Cr)
Potassium Iodate (59.3% I) .01 I .2
Ferric Citrate (21.2% Fe) 6 Fe 45
Manganous Carbonate (47.8% Mn) 3.5 Mn 59
Sodium Selenite (45.7% Se) .01 Se .16
Sucrose 119.93
Total 1000

a Mineral mix used in rat experiment, Chapter 6.










TABLE A-2. AIN-76A vitamin mix (As-fed)a.
Ingredient g IU/10 g

Vitamin A Palmitate (500,000 IU/g) .8 4,000
Vitamin D3 (100,000 IU/g) 1 1,000
Vitamin E Acetate (500 IU/g) 10 50


Menadione sodium Bisulfite (62.5%
Menadione)
Biotin (1.0%)
Folic Acid
Nicotinic Acid
Calcium Pantothenate
Pyridoxine-HCl
Riboflavin
Thiamin HC1


.08

2
.2
3
1.6
.7
.6
.6


mg/10 g
.5


.2
2
30
16
7
6
6
ug/10 g


Cyanocobalamin (.1%) 1 10
Sucrose 978.42
Total 1000

a Vitamin used in rat experiment, Chapter 6.














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