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The Effects of Zinc Supplementation
from Inorganic and Organic Sources in the
Diets of Neanling Pigs

presented by

Suzanne L. Hoover

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THE EFFECTS OF ZINC SUPPLEMENTATION FROM INORGANIC
AND ORGANIC SOURCES IN THE DIETS
0F WEANLING PIGS
By

Suzanne Lynne Hoover

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirments
for the degree of

MASTER OF SCIENCE

Department of Animal Science

1 997

ABSTRACT

THE EFFECTS OF ZINC SUPPLEMENTATION FROM INORGANIC AND
ORGANIC SOURCES IN THE DIETS OF WEANLING PIGS

By

Suzanne L. Hoover

Three experiments were conducted with weanling pigs tO determine the
efficacy Of zinc (Zn) supplementation from different forms during the nursery
phase. Growth performance, plasma and tissue accumulation Of Zn and Zn
balance were evaluated. Supplementation Of pharmacological concentrations Of
Zn in diets Of nursery pigs did not result in improved growth performance. Pigs
fed high Zn oxide (ZnO) had greater accumulation Of Zn in plasma, liver, kidney,
bone, urine and faces in all experiments (P<.05). In Exp. 2, pigs fed the control
diet were in a positive Zn balance and pigs consuming diets containing Zn
methionine complex, Zn amino acid complex and ZnO were in a negative Zn
balance after 15 d of supplementation. Results of these studies indicate that
nursery diets containing supplemental Zn at or above 500 ppm and fed for
greater than two weeks results in excretion of Zn in excess Of daily Zn

consumption.

ACKNOWLEDGMENTS

I would like tO express my deepest gratitude and respect tO my advisor Dr.
Gretchen M. Hill for her guidance and support in times when the light at the end
Of the tunnel seemed tO be absent. She has not only been my advisor but also a
dear friend that has shown me understanding, patience and belief in myself.

I extend my sincere appreciation to the other members Of my guidance
committee: Dr. Terry Ward for his endless support during my internship at
Consolidated Nutrition, LC. and also upon my return and throughout the
remainder Of my master’s program; Dr. Dale Rozeboom for his helpfulness with
my thesis; and Dr. Wanda Chenoweth for serving on my committee and kind
suggestions for improvements.

Many thanks to Jane Link for her helpful suggestions, patience and
friendship. Also thank you tO Marcia Carlson, Josep Garcia-Sierra and the rest
Of the swine department and farm crew for their help and friendship.

Most Of all I would like tO thank my parents for their unending support and
love during this endeavor. They have shared in my sorrow during times Of
misfortune but also have relished during times Of joy and achievement. Also
thank you to my friends for their emotional support throughout the completion of

my master's program.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................ v
LIST OF FIGURES ...................................................................................... vii
LIST OF ABBREVIATIONS ......................................................................... viii
INTRODUCTION ......................................................................................... 1
LITERATURE REVIEW ............................................................................... 5
Zinc Absorption ................................................................................ 5
Zinc Excretion ................................................................................... 9
Organic Forms Of Zinc ...................................................................... 11
Bioavailability ......................................................................... 12
Growth Performance .............................................................. 14
Immune Response ................................................................. 16
Link with Escherichia coli .................................................................. 17
CHAPTER 1

THE EFFECTS OF ZINC SUPPLEMENTATION FROM
INORGANIC AND ORGANIC SOURCES IN THE DIETS

OF WEANLING PIGS ................................................................................. 21
Abstract ............................................................................................ 21
Introduction ...................................................................................... 22
Materials and Methods ..................................................................... 24
Results ............................................................................................. 28
Discussion ........................................................................................ 34
Implications ...................................................................................... 39

APPENDIX A

THE EFFECTS OF ZINC SUPPLEMENTATION ON
METALLOTHIONEIN AND GUT MORPHOLOGY OF PIGS
INOCULATED WITH ESCHERICHIA COLI ................................................ 52

LITERATURE CITED .................................................................................. 59

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

LIST OF TABLES

Composition Of phase 1 basal diet in Exp. 1, 2, and 3 .......... 40

Composition of phase 2 basal diet in Exp. 1 and 3 ................ 41

Growth performance of weanling pigs fed supplemental
zinc from inorganic and organic forms (Exp. 1 and 2) ........... 42

Growth performance Of weanling pigs fed supplemental
zinc and inoculated with Escherichia coli (Exp. 3) ................. 43

Plasma mineral concentrations of weanling pigs fed
supplemental zinc from inorganic and organic forms
(Exp. 1 and 2) ........................................................................ 44

Tissue mineral concentrations Of weanling pigs fed
supplemental zinc from inorganic and organic forms

(Exp. 1) .................................................................................. 45

Tissue mineral concentrations of weanling pigs fed
supplemental zinc from inorganic and organic forms

(Exp. 2) .................................................................................. 46

Hepatic mineral concentrations Of weanling pigs fed
supplemental zinc and inoculated with Escherichia coli

(Exp. 3) .................................................................................. 47

Table 9. Urine and feces mineral concentrations of weanling
pigs fed supplemental zinc from inorganic and
organic forms (Exp. 2) ........................................................... 48

Table 10. Concentration Of metallothionein (MT) in the
intestine and liver of weanling pigs fed supplemental
zinc and inoculated with Escherichia coli (Exp. 3) ................. 57

Table 11. Morphological measurements of duodenal and
jejunal segments Of intestine from weanling pigs fed
supplemental zinc and inoculated with
Escherichia coli (Exp. 3) ........................................................ 58

vi

Figure 1.

Figure 2.

Figure 3.

Figure 4.

LIST OF FIGURES

Regulatory pathway Of dietary zinc processing by
intestinal cells ........................................................................ 8

Effect of zinc forms on zinc balance of nursery
pigs (d 16-20) ........................................................................ 49

Effect of zinc fonns on copper balance of nursery
pigs (d 16—20) ........................................................................ 50

Effect of zinc forms on iron balance Of nursery
pigs (d 16-20) ........................................................................ 51

vii

ADFI
ADG

DM

E. coli
et al.
G:F
min
MT
SEM
TGE

Tris

ZnMet
ZnO
ZnSO4

LIST OF ABBREVIATIONS

average daily feed intake
average daily gain

day

dry matter

Escherichia coli

et alia

gain to feed ratio

minute

metallothionein

standard error Of the mean
transmissible gastroenteritis
tris(hydroxymethyl)aminomethane
zinc amino acid complex
zinc methionine complex
zinc oxide

zinc sulfate

viii

INTRODUCTION:

Throughout the past decade the push toward increased production, as
well as confinement, has led to heightened concerns about the nutritional status
Of livestock. Since the animals do not come in contact with soil or pasture, there
is a greater potential of becoming deficient in one or more minerals (Pond,
1985). However, a mineral deficiency may be hard to discern. According to
McDowell (1992), “Mineral disorders can vary from acute deficiency with clinical
signs that are highly recognizable and pathological change, to mild conditions
that are hard to diagnose because Of the signs being decreases in
performance.”

Since, all living matter including feeds and body tissues is made up of
minerals, meeting the animal’s requirement is critical to maximize growth. The
importance Of minerals has been characterized by Underwood (1981 ), as stable
components of tissues and organs, constituents Of fluids and tissues in the
body, and within enzyme and hormone systems, minerals act as catalyst. The
skeleton consists of 80-85% Of the total body mineral matter, with calcium
comprising 46%, 29% phosphorus and together potassium, sulfur, sodium,

chlorine, and magnesium contribute 25%, and .3% is made up of trace minerals.

2

Even though essential trace elements only contribute .3% Of the total
body mineral, deficiency or excess of any mineral can lead to harmful effects.
Therefore, a greater concern on diet formulation with respect to minerals needs
to be emphasized. One such mineral is zinc (Zn). Although characterized as a
trace mineral, Zn has been used for thousands of years for practical purposes,
such as an Ointment for skin lesions, as well as for decorative and industrial
purposes. Zn is an essential trace element that is required for numerous
biological functions in all species Of livestock throughout all stages of the life
cycle. The essentialness of Zn for livestock was first reported in 1955 by Tucker
and Salmon, as it was shown to prevent parakeratosis in swine, a characteristic
of Zn deficiency disease.

Numerous other detrimental effects have been reported during Zn
deficiency, therefore the inclusion Of this trace element in the diets of livestock is
a common practice and has been for decades. In recent years the inclusion of
supplemental Zn above the requirement has been studied with results quite
variable. Several reports have shown that the use of pharmacological or high
concentrations Of Zn as zinc oxide (> 2000 ppm) in the diets of nursery pigs
improved growth (Hahn and Baker, 1993; Hill et al., 1996). On the contrary,
Fryer (1992) reported no significant improvement in growth performance with the
inclusion Of high concentrations Of Zn.

Initial reports from Europe linked this response to the suppression of

Escherichia coli (E. coir) proliferation and believed that in herds where

3

postweaning scours were a problem, supplementation of Zn would be
efficacious. Poulsen (1995) reported a decrease in the incidence Of non-specific
postweaning scours when pigs were fed pharmacological concentrations of ZnO
for the first two weeks of the nursery phase. However, the effect that Zn has on
pigs inoculated with E. coli has not been evaluated. At this physiological stage,
diarrhea in the weanling pig is increased because of the stress Of weaning and
the loss of maternal immunity which increases the pig’s susceptibility to
infectious bacteria (Sarrniento et al., 1988). This infectious diarrhea results in
substantial economic losses due to mortality, decreased growth rate, and costs
for treatment. Due to the suggested growth promoting effects and suppression
Of diarrhea, an increasing number of commercial nursery diets now contain high
concentrations of ZnO.

However, at these high concentrations, the amount of Zn excreted should
be evaluated and the impact on the environment questioned since excess Zn in
feces which in turn used as fertilizer could lead to soil accumulation and the
possibility Of plant toxicity. Ward et al. (1996) reported that the additional growth
response in the nursery phase due to high ZnO supplementation could also be
achieved with the supplementation of an organic form of Zn at a lower
concentration. Numerous studies have evaluated the significance Of organic Zn
versus inorganic forms, and the results are inconclusive. Therefore, our
Objectives were (1) to determine the efficacy of dietary Zn additions from

inorganic and organic forms of Zn on the growth performance Of nursery pigs,

4

(2) assess the excretion and retention Of different forms Of Zn at the
concentrations commonly used in the commercial industry, and (3) determine the
effect of supplemental Zn from inorganic and organic forms on the health and

growth Of weanling pigs inoculated with E. coli.

LITERATURE REVIEW

ZINC ABSORPTION:

Zn is absolutely essential for growth and development and to maintain
cellular functions. The means by which regulation of body Zn occurs are still
unclear and the regulatory roles that Zn absorption and excretion play within Zn
homeostasis are debatable. The mechanism by which Zn absorption occurs has
not been fully elicited and several theories exist. According to numerous studies
with the laboratory rat, Zn can be absorbed throughout the total length of the
small intestine. Various techniques in animal studies have yielded discrepant
results as to which region of the small intestine has the maximum absorptive
capacity. It has been postulated that the major site of absorption might shift with
Zn status. Conclusive evidence has been provided that intestinal Zn absorption
varies in response to an altered Zn supply status of the body (Flanagan et al.,
1983; Johnson et al., 1988). It has been regularly displayed that inverted sacs
from Zn deficient rats absorbed a greater amount of Zn than Zn supplemented
rats. The intestine itself has been suggested to be the major site for regulation.
Under normal conditions, absorption Of Zn is far below the limit Of saturation.
Three different stages of absorption occur and each step (alone and in

conjunction with the other steps) has been suggested to be the regulation point:

5

6

1) uptake from the lumen by cells lining the small intestine, 2) transport of Zn
through cells, and 3) discharge of Zn out Of basolateral membrane and into the
vascular compartment (Kirchgessner and Weigand, 1983).

Gastrointestinal digestion produces both high and low molecular weight
ligands and could affect Zn absorption by inhibiting or facilitating mucosal
uptake. Therefore, the mucosa would appear to be the major site of regulation
Of the absorptive process. Several theories exist as to the mechanism by which
Zn absorption is regulated. A majority Of the theories suggest that
metallothionein (MT) plays a key role in absorption. MT are cysteine rich
proteins that bind heavy metals; their synthesis is also stimulated by these
metals and other mechanisms such as stress (Andrews, 1990). MT binds
several metals including copper (Cu) and Zn. Each molecule Of MT binds 7
atoms of Zn(ll) and 12 atoms of Cu(l). Metallothionein has a high affinity for Cu
and can serve as a detoxification method. Hill et al. (1987) reported that oral Zn
could be used as a therapy in Wilson’s disease patients. This disease results in
excessive accumulation of copper at toxic levels in the brain and liver. They
concluded that oral Zn can be used as a therapy in decreasing the absorption of
Cu. Zn stimulated MT and Since MT preferentially binds Cu, excess Zn could be
used as therapy to stimulate MT and bind Cu. Since the turnover of the MT
complex is longer than that of the intestinal cells in which it is held, the bound Cu

and Zn are sloughed Off into the feces and excreted.

7
Nevertheless, with the potential Of MT to bind Zn, MT has been suggested

to play a role in absorption. Evans et al. (1975) reported that Zn absorption may
be linked with a low molecular weight ligand as well as metal free albumin and
receptor sites on the basolateral membrane. It was suggested that the Zn-
metallothionein (MT) complex in the intestine had a longer life compared to
turnover of villus cells and may reflect a regulatory mechanism for the disposal

in feces of that Zn remaining in mucosa. The synthesis Of MT in the intestine
also increased with an excess amount of Zn in the diet. In 1980, Starcher et al.,
reported that when mice were fed diets that were adequate in Zn, that absorption
was proportional to MT. On the contrary, Flanagan et al. (1983) concluded that
a difference in the amount of intestinal MT from Zn deficient and adequate rats
was found after five days of dietary treatment but not beyond five days. It was
suggested that the deficiency state progressed, and therefore stress induced MT
since differences were not seen after five days as would be expected since Zn
deficiency progressively worsens with time.

Richards and Cousins (1975) reported that when rats were parentally
loaded with Zn, serum concentrations initially increased and were followed by a
decrease which was concomitant with a greater accumulation of Zn in the liver
and also an increase in MT synthesis. From research completed by Richards
and Cousins (1976), intestinal MT appeared to not be a major regulator of
absorption and(or) binding component at all times. They suggested that when

Zn status was elevated by dietary or parental administration that MT synthesis

8

was enhanced. Wang et al. (1993) suggested that MT plays a limited role in the
regulation Of Zn absorption. When weanling pigs were fed high and adequate
Zn diets similar kinetic parameters were found and that the decrease in Zn
absorption when pigs were fed high Zn diets may be due to increased mucosal
passive diffusion of Zn rather than mediation of transport by a carrier. In 1979,
Cousins proposed a mechanism Of absorption shown in Figure 1. He concluded

that,

“A portion Of the dietary Zn which enters the lumen Of the small intestine
is transported across the mucosal brush border membrane. This transport
process probably requires ATP. The Zn content Of the intestinal cells which can
be changed by Zn status may influence the amount of Zn that is transported via
a carrier which is common to many divalent cations or uses a unique Zn
transport system. Isotope experiments have shown that with the intestinal cells,
newly acquired cytoplasmic Zn equilibriates with a Zn pool and is either shunted
into high molecular weight proteins and MT or transferred to the plasma. The
intracellular binding phase probably depends on rates of protein synthesis and
degradation and availability Of binding sites. In this regard MT may act as the
expandable (inducible) intracellular Zn binding compartment.”

 

 

LUMEN INTESTINAL CELL PL ASMA
DIETARY ZINC -
rm zinc \
mmmms
WWW
zinc cleum

 

EXCRETEO ZINC

 

 

 

 

Figure 1. Regulatory pathway of dietary zinc processing by Intestinal cells (Cousin. 1979).

9

These studies demonstrate that intestinal Zn absorption can respond rather
quickly to fluctuations in Zn supply and intestinal MT may serve as a temporary
storage protein for Zn. From MT, Zn may be passed through the vascular
compartment or returned to the lumen of the intestine. Therefore, the intestinal

wall may serve as the regulator of absorption and maintaining Zn homeostasis.

ZINC EXCRETION:

Zinc is excreted primarily in one of three ways: surface Of the skin, kidney
and the gastrointestinal tract. A majority of Zn is excreted in feces. Pekas
(1966) reported that pigs injected intravenously with labeled Zn excreted 75-90%
in feces. Unabsorbed Zn is the primary component Of fecal Zn; however,
endogenous sources also contribute a small amount. As stated previously, Zn
absorption varies depending on Zn status and intake. Excretion also follows a
similar pattern in that as Zn status increases, a greater amount Of Zn is excreted.
Johnson et al. (1988) reported that in the rat, the current diet affects Zn
absorption but, Zn of endogenous origin is not only affected by the current but
also by the past Zn intake.

Although endogenous Zn contributes a minor amount, it may play a role in
Zn homeostasis. Zinc accumulation in the lumen of the small intestine occurs

from several sources. Within the enterocytes, a bidirectional flux Of Zn may

10

occur and therefore deposit Zn back into the lumen. The pancreas and gall
bladder also secrete Zn into the lumen. As stated previously, the low-molecular
weight protein, MT, is synthesized via stimulation from excess Zn and has a
slower turnover rate than the enterocytes within which the complex is held.
Therefore, a large concentration Of Zn is deposited into the lumen via sloughing
of the epithelial cells. Weigand and Kirchgessner (1976) and Evans et al.
(1979) suggested that Zn homeostasis may be regulated by the amount that is
endogenously secreted back into the lumen and excreted in feces. Their
conclusions were derived from the isotope dilution technique which determined
the amount of Zn in the lumen that is of endogenous origin. It has also been
suggested that Zn secretions in the lumen from endogenous origin, could impact
absorption if intensive mixing Of unabsorbed and endogenously secreted Zn
occurs.

Excretion Of Zn can also occur in urine. This will reflectdietary treatment
and is thought to be from the plasma. Losses of Zn from body surfaces also
occur and are primarily in sweat (Forbes, 1983). Milne at al. (1983) reported
that in humans, the amount of Zn lost or excreted due to perspiration is directly
correlated with dietary intake. However, Zn loss can also occur due to sloughing

of skin and via hair, and in seminal fluid (Underwood, 1977).

1 1
ORGANIC FORMS OF ZINC:

In recent years much interest has developed in the area of organic trace
minerals. Several early varieties of organic metals have been studied
extensively such as EDTA chelates, proteinates, and metal polysaccharides.
However, metals complexed with amino acids are the focus of the current
discussion. The Association of American Feed Control Officials (AAFCO) has
provided definitions to distinguish the differences in organic forms of trace
minerals and are as follows:

57.151 Metal (Specific Amino Acid) Complex. The product resulting from
complexing a soluble metal salt with a specific amino acid.

57.150 Metal Amino Acid Complex. The product resulting from the
complexing Of a soluble metal salt with an amino acid(s).

57.142 Metal Amino Acid Chelate. The product resulting from the
reaction of a metal ion from a soluble metal salt with amino acids with a
mole ratio Of one mole of metal tO one to three (preferable two) moles Of
amino acid to form coordinate covalent bonds. The total molecular weight
of the hydrolyzed chelate must not exceed 800.

57.23 Metal Proteinate. The product resulting from the chelation of a
soluble salt with amino acids and/or partially hydrolyzed protein.

57.29 Metal Polysaccharide Complex. The product resulting form
complexing of a soluble salt with a polysaccharide solution declared as an
ingredient as the specific metal complex.

Numerous studies have evaluated the significance Of organic Zn forms

compared to inorganic, but the results are inconclusive. These studies span

across a wide range of spades including poultry, dogs, cattle, sheep and swine.

12

Zinc is routinely added to the diets Of animals, but the forms in which this mineral
is typically added are zinc oxide and zinc sulfate, which are inorganic forms. In
recent years there has been an increase in knowledge about the importance of
Zn in the diet and the added benefits Of high concentrations to weanling pigs. It
has also led to heightened interest in complexed forms Of Zn. Several studies
conducted have suggested that bioavailability of Zn is increased from organic
forms. Therefore the inclusion Of these products has become more prevalent in
commercial premixes for all species of livestock. Several theories have been
suggested with regards to organic forms and the mechanism by which they are
absorbed or metabolized. However, conclusive evidence has not been supplied

to elicit the answers.

5' 'l | TI

The most widely used theory for the use Of organic Zn forms is increased
bioavailability of Zn from organic forms. There are major discrepancies in the
literature with regards to the definition of bioavailability. Webster (1986) defines
bioavailability as the rate at which a drug, trace element, etc. enters the
bloodstream and is circulated to specific organs and tissues. However, O’Dell
(1984) suggests that the term bioavailability of a trace element refers to that
fraction Of the element in a food that is absorbed and utilized such as the

process Of transport, cellular assimilation and conversion to an active form.

13

As stated previously, research with organic forms of Zn spans across
many species. Pimental et al. (1991) completed two experiments with chicks
and they concluded that the bioavailability of Zn from Zn methionine and Zn
oxide were similar. These results were supported by a study done with isolated
rat duodenal segments (Hemp and Cousins, 1989) where Zn methionine and Zn
oxide were absorbed equally. However, several studies conducted using swine
as the animal model, concluded that Zn oxide was the least available form Of Zn
and that the intermediates were Zn methionine and Zn lysine. In these studies,
Zn sulfate proved to be the most available form of Zn when using parameters
such as bone, plasma and numerous other tissues that accumulate Zn (Schell
and Komegay, 1996; Wedekind et al., 1994). These findings were also
substantiated by experiments in other species. Nockels et al. ( 1993) concluded
that feeding calves diets containing Zn sulfate or Zn methionine resulted in
similar accumulation of Zn in plasma and tissues and therefore indicating that Zn
was not more available from either source. NO differences were reported by
Spears (1989) when lambs and heifers were fed Zn oxide and Zn methionine in
their diets when using plasma Zn and plasma alkaline phosphatase activity as
availability parameters. However, a lower excretion of Zn in urine was reported
for lambs fed Zn methionine. It was speculated that the difference in methionine
content of inorganic versus organic Zn forms could be the cause. On the
contrary, Rojas et al. (1995) concluded that lambs fed diets supplemented with

Zn in the forms of Zn lysine, Zn methionine, Zn sulfate or Zn oxide had

14

increased accumulation Of Zn in the liver, kidney and pancreas when fed Zn
lysine. In addition, these lambs fed Zn lysine, had increased metallothionein
concentrations in liver, kidney and pancreas compared to lambs fed the other
dietary treatments. However, Carlson et al. (1997) reported that a difference
was seen in the concentration of MT that was stimulated from organic and
inorganic forms of Zn. Organic forms of Zn fed to weanling pigs were found to
depress stimulation Of MT in the intestine, compared to weanling pigs fed the

control diet (Zn sulfate) or high Zn oxide supplemented diet.

GLQMLEerformame
Growth performance of animals fed diets supplemented with inorganic

versus organic Zn forms is as inconclusive as the information presented
previously on bioavailability. In a study conducted with hens and their progeny,
performance parameters were not different when fed diets with either Zn oxide of
Zn methionine (Kidd et al., 1992).

Several studies have shown that the addition of high concentrations of Zn
oxide can improve growth performance of weanling pigs (Hill et al., 1996; Hahn
and Baker, 1993). Although, the improvement in performance has been
variable, the supplementation of high Zn in nursery diets has become a common
practice. Many of the studies with organic forms Of Zn have suggested improved

bioavailability, therefore supplementation of these products has been tested to

15

determine if the enhanced performance from inorganic Zn could also be
achieved with supplementation of an organic form Of Zn at a lower concentration.
Ward et al. (1996) reported that supplementing nursery pig diets with Zn in the
form Of Zn methionine at 250 ppm enhanced growth performance equal to that of
2000 ppm Zn oxide. These results indicate that if the bioavailability of Zn from

' organic products is greater than Zn oxide than a lower concentration can be fed
and the improvement in performance can still be achieved. On the contrary,
Schell and Komegay (1996) reported no improvement in growth performance
from dietary additions of organic or inorganic forms Of Zn in the diets of weanling
pigs at three different concentrations (1000, 2000, or 3000 mglkg of diet). The
addition of pharmacological concentrations of Zn oxide in the diets Of nursery
pigs improved feed intake and weight gain; however, the inclusion of Zn
methionine or Zn lysine did not enhance performance above those fed the
control diet, when fed at the same concentrations as Zn oxide (Hahn and Baker,
1993). The growth performance Of heifers fed Zn methionine was not enhanced
over that Of heifers fed dietary treatments supplemented with inorganic Zn forms
(Spears, 1989).

In addition to performance, the quality of the products from livestock
supplemented with organic products has been evaluated. Kienholz et al. (1992)
reported that the inclusion of Zn methionine at one glkg of diet to laying hens
during a low calcium stress, maintained egg size and produced more eggs

(P < .05) during the stress period compared to hens fed no supplemental Zn

16

methionine or Zn methionine addition of two glkg of diet. Shell quality was also
evaluated and the results suggest that the addition Of Zn methionine in the diet
of laying hens receiving sodium chloride in the water at two gIL helped overcome
the negative effects of the sodium chloride and improved shell quality (Moreng et
al., 1992). Carcass characteristic for steers were improved with the addition Of
an organic forms of Zn in the diet. More specifically, Greene et al. (1988)
reported that steers fed Zn methionine diets had a higher quality grade and
greater amount of marbling. Although average daily gain and feed efficiency
were not affected by treatment, the increase in the previously mentioned carcass

parameters could prove to be beneficial economically.

ImmuneBesoonse
In addition to the previous suggested benefits Of organic Zn forms, it has

also been reported that these products may enhance immune response. Kidd et
al. (1992) reported that when chicks were fed Zn methionine in the diet, antibody
titers were significantly increased in chicks that had been challenged with
salmonella pullonem antigen. The addition of Zn methionine in the diets of
turkey poults helped clear a strain Of E. coli that had been administered to the
poults (Kidd et al., 1994). Similar improvements in immune response have also
been reported in cattle. Antibody titers against bovine herpesvr’rus-1 increased
in steers fed supplemental Zn methionine compared to control steers however,

in parainfluenza the antibody titers were not increased (Spears et al., 1991 ).

17

Steers challenged with infectious bovine rhino tracheitis virus and consuming
diets with Zn methionine had lower mean rectal temperatures and mean daily dry
matter intakes returned to pretrial levels sooner than control fed steers (Chirase

et al., 1991).

LINK WITH ESCHERICHIA COLI.

Mortality and reduced efficiency due to disease are costly to the swine
industry in achieving maximum production efficiency in the competitive US. and
world market. The area where great Opportunity to reduce costs for treatment is
in post-weaning scours. In the North-Central region of the United State, Crooks
et al. (1993) reported that the increased profit potential due to a 10% reduction
in scours would be $39 to $434 depending on herd size. Hurley et al. ( 1995)
reported that the incidence of scours in pigs is significantly greater in the
Midwest than the Southeast region. These losses cost Midwestern producers
$2.05 per pig compared to $0.62 in the Southeast. Relative to the weanling pig,
16.11% Of the pigs up to 40 pounds, show signs Of scouring.

The growth response associated with pharmacological concentration of
Zn in the diets Of nursery pigs maybe linked to the suppression Of E. coli.
Poulsen (1995) reported a decrease in the incidence Of non-specific scours
when pigs were fed pharmacological concentrations of zinc oxide for the first two
weeks for the nursery phase. It was reported by Kidd et al. (1994) that turkey

poults administered E. coli and fed a Zn methionine supplemented diet, cleared

18

the strain at a faster rate than control or inorganic supplemented diets.

As noted by Holland (1990), bacteria cause diarrhea by (1) producing
enterotoxins that influence the crypt cells to hyper secrete, (2) eliciting an
inflammatory response from the invasion Of the intestinal mucosa and (3)
destroying the absorptive capacity Of the villi resulting in malabsorption.
Escherichia coli is a gram-negative bacteria and is an anaerobic, non-spore
forming member of the Enterobacteriaceae family. It causes a profuse and
watery diarrhea with dehydration of up to 10 to 12% loss of body weight in a six
hourpeNOd.

The importance Of enterotoxigenic E. coli as the cause Of diarrhea among
young farm animals is well recognized. Escherichia coli is a common inhabitant
of the intestinal tract of humans and animals, but some strains cause infections
and diarrhea in young farm animals. This bacteria may act alone or in
conjunction with other bacteria such as rotavirus (Holland, 1990).

The family Of Enterobacten’aceae is made up of a series of interrelated
bacterial types. Each member of the family has an endotoxin which is a
lipopolysaccharide complex in the outer membrane and contains an O-specific
serologic group. Additionally, some members, especially E. coli, produce
enterotoxins. Escherichia coli produce two enterotoxins a heat-labile and a heat-
stable. The family is further divided into subgroups on the basis of serology of
the O antigens of the bacteria. The 0 antigen subgroups are divided into

serotype, which are characterized by the possession of particular 0 antigen

19

fractions, K antigens or H antigens. The K antigen denotes polysaccharide
capsular antigens. The K antigen is further divided into three different types
according to physical characteristics: L, A, and B (Sojka, 1965). According to
Holland (1990) and Wilson (1986) the most common cause Of diarrhea in pigs is
from the enterotoxigenic E. coli K88 strain, which has been reported to cause
diarrhea in pigs pre and postweaning. Sarrniento et al. (1988) reported a
reproducible model of postweaning scours caused by enterotoxigenic E. coli
utilizing antigen K88.

The pathogenesis of the E. coli is initiated by the adherence of the
bacteria to the epithelia of the small intestine. The colonization is mediated by
appendages called pill or fimbriae that adhere to the intestinal mucosa. More
specifically, on the tip Of the fimbriae is a protein, called adhesin, which is the
major factor in adherence to the intestine. Some animals may be genetically
resistant to some strains of E. coli. In order for the bacteria to colonize the
intestine of animal species, the villous epithelium must have receptors for the
fimbriae (Nagy et al., 1992). If the receptor is not present on the epithelial cell,
the animal will be resistant to that strain of E. coli. The presence Of the receptor
is genetically mediated. Age—related resistance to other strains of E. coli such as
987P+ strains have been reported by Dean-Nystrom and Samuel (1994).
Neonatal pigs less than six days of age were susceptible to colonization of the
small intestine by the 987 strain but, weaned pigs were resistant. Katouli et al.

(1995) reported phenotypical characteristics of intestinal E. coli of pigs during

20
suckling, postweaning and finishing periods. They concluded that the

colonization and persistence of E. coli in pigs is dependent on both the strain
and host specificity.

Weaning has been suggested to reduce resistance to infection because
Of associated circumstances such as stress. These factors include the loss Of
maternal contact, introduction into strange pens and pen-mates, and the loss Of
maternal immunity from milk. Hampson (1986) reported that weaned pigs, in
contrast to unweaned pigs, experienced alterations in intestinal architecture after
weaning. Changes included dramatic increases in crypt depth and an increase
in the complexity of villus morphology with a large reduction in villus height. He
concluded that due to the changes in morphology primarily with the absorptive
area of the small intestine, susceptibility Of piglets to infection or bacterial
invasion may be increased. Opportunistic bacteria are given a chance to invade
since the pigs immune system may be compromised due to these factors.
Consequently, infectious diarrhea may occur and result in substantial economic
losses due to mortality, decreased growth rate and cost for treatment. However,
Sanniento et al. (1988) reported that weaning itself is not required tO induce

diarrhea but may increase the severity Of the scouring.

THE EFFECTS OF ZINC SUPPLEMENTATION FROM
INORGANIC AND ORGANIC SOURCES IN THE DIETS OF
WEANLING PIGS

ABSTRACT

Three experiments were conducted with weanling pigs to determine the
efficacy of zinc (Zn) supplementation from different forms of Zn during the
nursery phase. In Exp. 1, 172 PIC pigs were weaned (average 19 d and 6.47
kg) and allotted to dietary treatments for a total Of 33 d: basal (control), basal +
250 ppm zinc methionine complex (ZnMet), basal + 500 ppm zinc oxide (ZnO),
basal + 250 ppm zinc amino acid complex (ZnAA), basal + 500 ppm ZnAA, basal
+ 250 ppm ZnAA + 500 ppm ZnO, and basal + 3000 ppm ZnO. In Exp. 2, 12 PIC
barrows (average 19 d and 6.68 kg) were allotted to a dietary treatment: basal
(control), basal + 250 ppm ZnMet, basal + 250 ppm ZnAA, and basal + 3000
ppm ZnO. Pigs were housed in individual stainless steel metabolism cages for a
20 d trial. Feces, orts and urine were collected for 5 d following a 15 d
adjustment period. In Exp. 3, 72 crossbred pigs were weaned (average 16 d and
4.86 kg) and allotted to a 28 d trial with a 3 x 2 factorial arrangement of
treatments. Each dietary treatment was represented by three replicates of

Escherichia coli (E. coir) inoculated and three sham inoculated pigs. Dietary

21

22
treatments were: basal (control), basal + 250 ppm ZnAA, and basal + 3000 ppm

ZnO. Blood samples were obtained on d 0 and 33 Of Exp. 1 and d 0 and 20 of
Exp. 2. On d 33 Of Exp. 1, two pigs per replicate (n=70), on d 20 of Exp. 2 and d
28 of Exp. 3, all pigs were killed and tissues collected. Supplementation of
pharmacological concentrations of Zn in the diets of nursery pigs did not result in
improved growth performance in Exp. 1 or 3. Pigs fed ZnO, had greater
accumulation Of Zn in plasma, liver, kidney, bone, urine and feces in all
experiments (P<.05). In Exp. 2, pigs fed the control diet were in a positive Zn
balance, and pigs consuming diets containing ZnMet, ZnAA and ZnO were in a
negative Zn balance. Results of these studies indicate that nursery diets
containing supplemental Zn at or above 500 ppm and fed for greater than two

weeks results in excretion of Zn in excess Of daily Zn consumption.

Key words: Pigs, Zinc, Tissues, Plasma

INTRODUCTION

The use of pharmacological concentrations of Zn as ZnO in the diets of
nursery pigs has been reported to have growth promoting properties (Hahn and
Baker, 1993; Hill et al., 1996). Initial reports from Europe linked this response to

the suppression of E. coli proliferation. Pigs at weaning have an increased

23

susceptibility to diarrhea due to opportunistic infection during stress (Sarrniento
et al., 1988). This infectious diarrhea results in substantial economic losses due
to mortality, decreased growth rate, and costs for treatment. Poulsen (1995)
reported a decrease in the incidence of non-specific post-weaning scours when
pigs were fed pharmacological concentrations of ZnO for the first two weeks Of
the nursery phase.

Due to the suggested growth promoting effects and suppression Of
diarrhea, nursery diets now commonly contain high concentrations Of ZnO.
However, at these high concentrations, the amount of Zn excreted should be
evaluated and the impact on the environment questioned. Ward et al. (1996)
reported that the additional growth response in the nursery phase
due to high ZnO supplementation could also be achieved with the
supplementation of an organic form of Zn at a lower concentration. Numerous
studies have evaluated the significance of organic Zn versus inorganic forms
and the results are inconclusive. Therefore, our objectives were (1) to determine
the efficacy of dietary Zn additions from inorganic and organic forms of Zn on the
growth performance of nursery pigs, (2) assess the excretion and retention of
different forms Of Zn at the concentrations commonly used in nursery diets
today, and (3) determine the effect Of supplemental Zn from inorganic and

organic forms on the health and growth of weanling pigs inoculated with E. coli.

24
MATERIALS AND METHODS

All experiments were approved by the Michigan State University Animal
Care and Use Committee prior to initiation of the study. Exp. 1 and 2 were
completed at Consolidated Nutrition Research Center, Decatur, IN., and Exp. 3
was completed at Michigan State University.

Experiment 1. One hundred sixty-seven pigs of Pig Improvement
Company (PIC) genetics (Line 326 X C22) were weaned at 19 d of age and 6.47
kg, and used in a randomized complete block experiment. Pigs were allotted to
one of seven dietary treatments: basal (control), basal + 250 ppm Zn as ZnMet,
basal + 500 ppm Zn as ZnO, basal + 250 ppm Zn as ZnAA, basal + 500 ppm Zn
as ZnAA, basal + 250 ppm Zn as ZnAA + 500 ppm Zn as ZnO, and basal + 3000
ppm Zn as ZnO. Phase 1 diets were fed from d 0-14, with the basal diet
containing 250 ppm calculated Zn as zinc sulfate (ZnSO,), and 100 ppm
calculated copper (Cu) as copper lysine and 25 ppm Cu as copper sulfate
(CuSO,,). Phase 2 diets were fed from d 14-33, and the basal diet contained 160
ppm calculated Zn as ZnSO4 and 50 ppm calculated Cu as copper lysine and 16
ppm Cu as CuSO4. All diets (Table 1 and Table 2) met or exceeded NRC
nutrient recommendations (NRC, 1988) for weanling pigs. Four or five pigs were
allotted by sex and litter to a dietary treatment with five weight replicates. Pigs

were weighed on d 0, 14, and 33.

25
Experiment 2. Twelve PIC barrows (Line 326 x 022) were weaned (19 d

Of age and 6.68 kg) and allotted by litter to one of four dietary treatments as
follows: basal (control), basal + 250 ppm Zn as ZnMet, basal + 250 Zn as ZnAA,
and basal + 3000 ppm Zn as ZnO. All diets (Table 1) met or exceeded NRC
nutrient recommendations for weanling pigs (NRC, 1988). Basal diet was the
same as the phase 1 basal diet in Exp. 1. Pigs were assigned to individual
stainless steel pens equipped with stainless steel feeders and waters in a
temperature controlled room. For the first 14 d, pigs were given ad libitum
access to feed and distilled water. On d 15, pigs were fed 90% of daily ad
libitum intake divided into three feedings per day. This regime was followed for
the next 5 d and weigh backs were recorded as necessary. Feces, orts and
urine were collected for five, 24 h periods. Pigs were weighed daily for the first
15 d and every other day thereafter. ‘

Experiment 3. Seventy-two crossbred pigs (Duroc x (Yorkshire x
Landrace)), having received no creep feed prior to weaning, were weaned at an
average age of 16 d and average weight of 4.86 kg and allotted by sex and litter
to a 28 d trial with a 3 x 2 factorial arrangement of treatments. Six separate
temperature controlled rooms were used with three pens per room and four pigs
per pen with ad libitum access to feed and water. Each dietary treatment was
represented by three replicates of E. coli inoculated and sham inoculated pigs.
The dietary treatments across the two health status were as follows: basal

(control), basal + 250 ppm Zn as ZnAA, and basal + 3000 ppm Zn as ZnO.

'- J-r-n-v

26
Phase 1 (d 0-14) and phase 2 (d 14-28) basal diets were the same as those in

Exp. 1. All diets (Table 1 and Table 2) were pelleted and met or exceeded NRC
nutrient recommendations (NRC, 1988). Individual pigs were weighed on d 0, 7,
14, 21, and 28 post-weaning and feed disappearance was recorded.

At weaning, three rooms Of pigs received an oral dose Of 5 mL of
enterotoxigenic Escherichia coli (serotype 01492k88 + non motile) inoculum (1.3
x 10"° CF UlmL), in peptone saline. A sham of peptone saline was orally
administered to pigs in the remaining three rooms. On d 5, pigs that had
previously received E. coli were given another 5 mL Of E. coli inoculum
(3.2 x 1010 CFUImL). On d 7, one pig per replicate group (n=18) was killed.

The remaining pigs (n=54) continued on test for 21 d. Pigs were Observed daily
for evidence of diarrhea. Scour scoring was done by an individual who was
blind to the study protocol. Rooms inoculated with sham were scored first and
then rooms inoculated with E. coli to avoid cross-contamination. The index used
for scoring was as follows: 1=no loose stools in pen, 2=one-half of the stools in
pen are loose, 3=all stools in pan are loose, 4=one-half Of stools in pan are
watery and one-half loose, and 5=all stools are very watery.

Plasma. Blood samples for all pigs were taken in Exp. 1 on d 0 and 32
and in Exp. 2 on d 0 and 20, from the anterior vena cava into heparinized
vacutainer tubes and placed on ice. Plasma was separated by centrifugation at
3000 x g for 15 min at 5°C and stored at -80°C until analyzed for Cu, Fe, and Zn.

Mineral concentrations in plasma were determined by flame atomic absorption

27
spectrophotometry (Smith-Hieftje 4000, Them'lo Jarrell Ash Corporation,

Franklin, MA) after a 1:7 dilution with deionized distilled water.

Tissues. In Exp. 1, d 33, two pigs per replicate and on d 20 of Exp. 2, all
12 pigs, were killed by electric-immobilization and exsanguination. Fifty-four
pigs in Exp. 3, were killed by injecting penibarbitol (1 mU4.5 kg Of BW) into the
jugular vein. From pigs in Exp. 3, the liver and several segments of the intestine
were removed. Further description Of methods used for Obtaining intestinal
segments is listed in Appendix A. Liver, left kidney and right front foot were
removed from all pigs in Exp. 1 and 2 and a portion of the longissimus muscle
from pigs in Exp. 2. Samples were immediately weighed and then placed on ice.
Dry matter of liver and kidney were determined separately by drying in vacuum
oven (Heinicke, Portland, OR). Frozen metacarpal bones were cleaned of all
adhering tissue and thawed before being weighed and dry ashed in muffle oven
(BamsteadfThermolyne, Dubuque, IA). Bone ash was weighed and digested
with 30 mL of 10 M nitric acid. Longissimus muscle samples were freeze dried
(Vitris 25 SRC, The Vitris Company, Gardiner, NY) for dry matter determination
and then ground in a stainless steel blender. Tissue samples were prepared for
mineral analysis by nitric-perchloric acid wet digestion (AOAC, 1990) modified
using 3 mL of 10 M perchloric acid and 20 mL Of 10 M nitric acid. Mineral
concentrations were determined by flame atomic absorption spectrophotometry.

Urine, Feces, and Feed. In Exp. 2, urine was collected twice every 24 h

period, pooled, mixed and two aliquots were frozen at -80°C for later mineral

28

analysis. Feces were collected twice every 24 h period, weighed and also
stored at -80°C. Fecal samples were freeze dried for determination of dry matter
content then ground in stainless steel blender. Feces and feed samples were
prepared for mineral analysis according to nitric-perchloric acid wet digestion,
using 3 mL of 10 M perchloric acid and 20 mL Of 10 M nitric acid. Mineral
concentration of feed, feces and urine were determined by flame atomic
absorption spectrophotometry.

Statistical Analysis. Data for the three experiments were analyzed using
GLM procedures and least squares Of means by treatment (SAS, 1995). Some
Of the data were log transformed to increase normal distribution after detection
Of heterogeneity Of variance problem using a modified Levene’s test. The mean
differences between the treatments were detected by comparison Of least square
means. Differences were considered significant at P < .05. Several preplanned
comparisons were used when analyzing data from Exp. 1 and they are as
follows: ZnMet vs. ZnAA, 250 ZnAA, 500 ZnO, 250 ZnAA + 500 ZnO effect and a

linear and quadratic effect for the ZnAA dietary treatment.

RESULTS

Growth Performance. The effects of source and concentration of
supplemental Zn on average daily gain (ADG), average daily feed intake (ADFI),

and gain to feed ratio (GF) are summarized in Table 3. From d 0 to 14 in Exp. 1,

29
ADG Of pigs fed the diet with 3000 ppm supplemental Zn as ZnO was lower

(P < .05) than that of pigs fed the diets supplemented with Zn at 500 ppm ZnO,
250 ppm Zn as ZnAA, or 250 ppm Zn as ZnAA + 500 ppm Zn as ZnO. Similar
results were achieved for ADFI with those pigs fed 3000 ppm Zn as ZnO,
consuming less feed per day during phase 1. Overall, pigs fed diets containing
an additional 500 ppm Zn as ZnO tended to gain faster and consume more feed
than those fed the other diets from d 0 to 14. Due to the restricted feed intake of
pigs in Exp. 2 for the 5 d collection period, the weight gains and feed
consumption Of these pigs are not discussed however, growth performance data
are presented in Table 3. All growth performance parameters measured in Exp.
3 (Table 4) were not statistically different between dietary treatments or across
the two health statuses (P > .05).

Plasma. The effects of source and concentration of supplemental Zn on
plasma minerals of pigs in Exp. 1 and 2 are listed in Table 5. In Exp. 1, plasma
Zn concentration was greater (P < .05) from pigs fed the 3000 ppm ZnO.
Plasma Zn concentration of pigs fed 250 ZnAA + 500 ZnO diet was not different
than the plasma Zn concentration of pigs fed 250 ZnMet, 500 ppm ZnO, 250
ZnAA, or 500 ZnAA. The lowest plasma Cu concentrations were seen when pigs
were fed the control diet or 500 ppm ZnO but not 3000 ppm ZnO which would be
expected.

In Exp. 2 there were no differences in plasma Zn between pigs fed the

control diet or diets containing ZnMet or ZnAA even though, the concentration of

30

Zn in the organic supplemented diets was calculated to contain 250 ppm more
Zn than in the control diet. However, pigs fed the ZnO diet had greater
concentrations of Zn in plasma on d 20 (P=0.0001). Plasma Cu and Fe
concentrations were not different between dietary treatments (P > .05).

Organ weights. In Exp. 1 (data not shown), the kidney weight as a
percentage of body weight was increased ( P < .05 ) in pigs fed 250 ZnAA
compared to those fed control diet, 250 ZnMet, or 250 ZnAA + 500 ZnO diet.
There was also a ZnMet versus ZnAA , 250 ZnAA + 500 ZnO effect, and ZnAA
quadratic effect (P<.05), for kidney weight as a percentage Of body weight.

Tissues. The effects Of source and concentration of supplemental Zn on
the Zn, Cu and Fe concentrations of tissues are presented in Table 6, 7, and 8.
The percent dry matters of all tissues analyzed in Exp. 1, 2, and 3 were not
statistically different between dietary treatments. Hepatic and renal Zn
concentrations were greater from pigs fed the 3000 ZnO diet than pigs fed all
other dietary treatments (P = .0001 ). Liver Cu was not different between dietary
treatments (P > .05) however, renal Cu concentration was increased when pigs
were fed 3000 ppm supplemental ZnO. Hepatic and renal Fe were elevated
from pigs fed the 250 ZnAA diet. Pigs fed 250 ZnAA had greater renal Fe
concentrations compared to control, 250 ZnMet, 500 ZnO, 250 ZnAA + 500 ZnO
pigs at P<.05.

Zn concentrations in the metacarpal bone (Table 6) followed the same

pattern as plasma, liver and kidney Zn, in that the pigs fed the high Zn oxide

31

(3000 ppm) retained more Zn in their metacarpal bone than pigs fed all other
dietary treatments (P<.0001). Bone Cu was not different due to dietary
treatment however, bone Fe was decreased in the bones of pigs consuming the
control diet compared to pigs fed 3000 ZnO (P<.05).

In Exp. 2, liver Zn (Table 7) was greater in those pigs fed ZnO than those
consuming the control diets or diets containing either of the organic Zn products
(P=0.0001). Zn concentrations in the liver were not significantly different
between pigs fed diets containing control, ZnMet or ZnAA. The concentrations
of hepatic Cu and Fe were not different between dietary treatments. Renal Zn
and Cu were greater in those pigs fed ZnO however no differences in kidney Fe
were seen. NO significant differences in Cu, Fe or Zn concentrations in muscle
were seen between dietary treatments. Pigs fed ZnMet appear to be depositing
a greater amount of Zn in muscle tissue compared to pigs fed the other dietary
treatments however, they are only numerically and not different. There was a
trend for greater Cu concentration in muscle when pigs were fed ZnMet although
not significant. The Fe and Cu concentrations in bone were not different
between dietary treatments. Pigs fed the diet containing ZnO, retained a greater
amount Of Zn in bone than pigs fed control, ZnMet, or ZnAA diets, (P=0.0001).

In Exp. 3, (Table 8) the hepatic Zn, Cu and Fe concentrations were not
different between pigs fed the control or ZnAA supplemented diets with or
without E. coli or sham inoculation. However, liver Zn was elevated ( P < .05 )in

ZnO fed pigs (E. coli and sham inoculated), compared to control and ZnAA fed

32

pigs. E. coli inoculated pigs fed ZnO had decreased Zn concentration in liver
compared to sham inoculated pigs within the same dietary treatment.

Scour Scoring. In Exp. 3 (data not shown), scores were assigned to each
pen to determine the effect of Zn supplementation on scouring. The most

prominent differences in scour scores were seen from d 4 to d 7. On d 4, pigs

" ' '.".'£ _ "-.- m '

that were inoculated with E. coli and were consuming the ZnAA and ZnO
supplemented diets had looser stools than pigs fed the control , ZnAA and ZnO
supplemented diets and inoculated with sham ( P < .05). Similar results were
seen on d 5 however, pigs fed the control diet and inoculated with E. coli had
looser stools than control sham inoculated pigs ( P < .05). On d 6, again E. coli
inoculated pigs had looser stools across all dietary treatments than pigs given
sham inoculation. Pigs receiving the ZnAA supplemented diet and inoculated
with the sham had looser stools than control sham inoculated pigs (P < .05). On
d 7, stools followed the same pattern with E. coli inoculated pigs with looser
stools however, the pigs with the finnest stools were those consuming the ZnO
diet and inoculated with sham. After d 7, stools were at a similar consistency
until d 19 when pigs consuming the ZnAA supplemented diet and inoculated with
sham had the firrnest stools. Pigs consuming the ZnO supplemented diet and
inoculated with sham exhibited the finnest feces on d 21, 22, 25 and 26.

Urine and Feces Mineral Concentrations and Balance. The effects of
supplemental Zn source and concentration on excretory products and balance

are given in Table 9, and Figures 2, 3, and 4. As Zn increased in the diet

33

regardless Of form, urinary and fecal Zn increased. Pigs that were fed the
dietary treatment containing 3,000 ppm supplemental ZnO had increased Zn
concentration in urine and feces compared to pigs fed the control diet, ZnMet or
ZnAA supplemented diets ( P < .05). Fecal Cu was not different between dietary
treatments. The greatest concentration of Fe that was excreted in the feces was
from pigs fed diets containing supplemental Zn in the form of ZnMet and ZnO.
Pigs fed the supplemental Zn as ZnAA were excreting greater amounts of Zn
and Cu in urine than pigs on control diet. Urinary Cu from pigs fed control and
ZnMet was less than pigs fed ZnAA or ZnO (P < .05). Contrast to urinary Zn
and Cu excretion from pigs fed ZnMet or ZnAA products in the diet, pigs fed
ZnMet excreted a greater amount of Fe in feces compared to pigs fed ZnAA,
however this was not significant (157 mg vs. 133 mg Of Zn in feces from pigs fed
ZnMet or ZnAA, respectively). Urinary excretion of Fe was greatest for ZnAA
pigs but only different from control and ZnO fed pigs (P<.05).

Although, data suggest trends for differences in Zn balance (Figure 2)
between dietary treatments, differences were not significant. The only pigs in
positive balance were those fed the control diet. However, Zn balance was
negative for all other dietary treatment meaning more Zn was excreted than
taken in each day after receiving the respective diet for 15 d prior to initiation Of
balance. Pigs fed the control and ZnAA diets were in a positive balance for Cu
(Figure 3) unlike ZnMet and ZnO fed pigs. However, Fe balance (Figure 4) was

positive for pigs fed all dietary treatments.

 

DISCUSSION

In this study, feeding supplemental Zn in the diets Of weanling pigs
resulted in variable performance and are similar tO findings reported by Fryer et
al. ( 1992) and Schell and Komegay (1996). The increased growth performance
due to the supplementation of high concentrations of ZnO reported by Hahn and
Baker (1993) and Hill et al. (1996) were not seen in this study. The results of
Exp. 1 show a decrease in performance especially during the first two weeks of
the nursery phase when pigs were fed diets containing 3000 ppm Zn as ZnO
compared to other dietary treatments. Numerous studies have been completed
to test the hypothesis that high concentrations Of Zn in the diet of nursery pigs
improves growth performance also indicating inconsistent performance.

It has been speculated that health status Of the pigs plays a key role.
Poulsen (1995) reported that the addition Of high concentrations Of ZnO in the
diet of nursery pigs for two weeks reduced the incidence of diarrhea. It has
been postulated that whether the pigs are suffering from post-weaning scours
influences the effect of high zinc in the diet. However, in Exp. 3 pigs inoculated
with E. coli and(or) sham did not grow differently relative to Zn concentration or
form in the diet. Growth performance parameters measured were not different
between dietary treatments or between pigs inoculated with sham or E. coli.

Health status may not be the only influence in whether positive or negative

35

results are seen due to Zn supplementation. Factors such as age and sanitation
of the facility in which the pigs are housed may have an impact. Pigs in Exp. 3
were housed in a pristine facility built specifically for research with infectious
bacteria. This facility is new and the sanitation is impeccable compared to on-
farm trials. The ability of additional bacteria to proliferate was minimal if not void
since rooms and pens were washed and sanitized daily. Due to the strict
sanitation of the facility and the inclusion of an antibiotic in the diet, results of
this study may differ from on-fann trials.

Ward et al. (1996) reported that the additional growth response in the
nursery phase due to high ZnO supplementation could also be achieved with the
addition of zinc methionine at a lower concentration. In Exp. 1, pigs receiving
diets containing 250 ppm ZnAA or 250 ppm ZnAA + 500 ppm ZnO had improved
growth performance above that achieved by pigs supplemented with 3000 ppm
ZnO during phase 1 (P < .05).

The concentration of Zn in plasma, liver, kidney, bone , urine and faces
were greater ( P < .05) from pigs fed the high ZnO diets than from pigs fed all
other dietary treatments in Exp. 1, 2, and 3. It has been postulated that the
importance Of the chelated or complexed minerals is the increased
bioavailability. While tissue storage is not the same as bioavailability, there were
minimal differences in tissue Zn concentrations between dietary treatments
containing no supplemental Zn or supplemental Zn in the form of ZnMet or ZnAA

in Exp. 2. Results of Exp. 2, show that after the 15-d adjustment period, pigs

36

receiving the control diet were in a positive Zn balance, therefore indicating that
they were retaining more Zn than what they were taking in on a daily basis.
However, pigs receiving diets containing supplemental Zn from ZnMet, ZnAA or
ZnO were excreting more Zn per day than these pigs were consuming during the
third week of dietary intervention. When comparing plasma, liver, kidney and
bone Zn concentrations over the three studies, it is evident that metabolism of
Zn must change after several weeks of consuming a diet containing
supplemental Zn above the requirement for the weanling pig. The concentration
Of Zn in the basal diet of pigs in Exp. 1 during phase two (d 14-33) was only
calculated to be 90 ppm less than what pigs in the Exp. 2 were receiving for 20
d. However, plasma Zn concentration in pigs from Exp. 1, were almost half as
much as plasma Zn on d 20 of Exp. 2. Hepatic Zn concentrations also followed
this same trend. For example, from data in Table 6, 7, and 8, the hepatic Zn
concentration Of pigs in Exp. 2 on d 20 that were fed 3000 ZnO was 1166.22
rig/g Of wet tissue but, the liver Zn concentration of pigs fed 3000 ppm ZnO in
Exp. 1 on d 33 was 580.79 uglg and in Exp. 3 the sham inoculated pigs fed ZnO
had hepatic Zn concentration on d 28 Of611.35 uglg. These differences were
also seen in kidney and bone and for dietary treatments that were similar across
all three studies including 250 ZnMet, 250 ZnAA and 3000 ZnO. These results
indicate transitory changes in Zn concentrations in body compartments relative
to age and Zn source and concentration. It has been reported that Zn

absorption is affected by Zn status and that as dietary Zn increases absorption

37

decreases in pigs and rats (Wang et al., 1993; Jackson et al., 1981) Therefore,
by the third week, the accumulation Of Zn in the plasma and tissues may have
reached a threshold and the ability to absorb Zn is decreased. A greater amount
of dietary Zn is not absorbed but is excreted via feces. Although, not evaluated
in this study, it is likely that at this time, the amount of endogenously secreted Zn
may also increase. Pekas (1966) reported that fecal Zn Of pigs on an adequate
Zn intake included unabsorbed as well as endogenously secreted Zn. Our work
demonstrates that supplementation of Zn in the diets of weanling pigs at or
above 500 ppm from inorganic and organic sources of Zn during the third week
of the nursery phase results in excess Zn excreted in urine and feces. The pigs
consuming ZnAA, ZnMet Of ZnO supplemented diets during the third week of
Exp. 2 were in a negative Zn balance and therefore excreted more Zn than what
they were consuming on a daily basis (Figure 2). Supplementation during this
time period appears to not play a role in enhancing performance.

Carlson et al. (1996) reported that feeding pharmacological
concentrations Of Zn in the diets of nursery pigs for the first two weeks improved
growth performance as much as four weeks Of supplementation. Our results
would indicate that after two weeks, excess Zn is excreted and therefore
supplementation should only be for two weeks. Plasma and tissue data would
also support this statement due to the apparent mobilization of Zn stores during
the 3 and 4 wk of the nursery phase to possibly rid the body of excess Zn stores.

In Exp. 3, hepatic Zn concentrations of ZnO fed pigs inoculated with E.

38

coli were decreased compared to sham inoculated pigs which is in contrast to
reports of increased hepatic Zn concentration and decreased plasma Zn in
human during infection and in chicks inoculated with E. coli (Tufft et al., 1988).
This was also supported by findings from Whitenack et al. (1978) that pigs fed
adequate Zn diets and infected with transmissible gastroenteritis had decreased
plasma Zn concentrations and increased hepatic storage of Zn. In Exp. 2, pigs
consuming the high ZnO diet had decreased hepatic Cu concentrations and
increased renal Cu. The results are supported by finding by Hill et al. (1983)
that reported decreased liver Cu stores when high dietary Zn was fed.

The Fe and Cu concentrations in bone were not different between dietary
treatments in Exp. 2. Pigs fed the diet containing ZnO, retained a greater
amount of Zn in bone than pigs fed control, ZnMet, or ZnAA diets, (P=0.0001).
Our data suggests that the concentration of Zn in the bone was not different
between pigs fed the control, ZnMet or ZnAA diets even though diets containing
organic products were formulated to contain 250 ppm more supplemental Zn
than the control diet. Actually, pigs consuming diets containing organic
products numerically retained less zinc in bone than the control diet, although
not significant (1026.35 #919, 984.65 uglg, and 969.30 uglg Of zinc in bone from
pigs fed control, ZnMet, or ZnAA, respectively).

Zinc concentration in muscle tissue in Exp. 2 were not different between
dietary treatments. Schell and Komegay (1996) reported that feeding

pharmacological concentrations of ZnO to nursery pigs for 2 wk resulted in lower

39

concentration of Zn in muscle compared to pigs fed the control diet at 105

mglkg.

IMPLICATIONS

The results Of this study show that supplementation Of Zn at or above 500
ppm from inorganic and organic sources during wk 3 of the nursery phase led to
excess excretion Of Zn beyond amount consumed. Supplementation during the
3 wk may lead to alterations in Zn metabolism by decreasing Zn absorption and
ridding body of excess Zn. However the environmental impact Of this excessive

Zn in waste is unknown and warrants further research.

Table 1. Composition Of Phase 1 (d 0-14 for Exp. 1 8 3
and d 0—20 for Exp. 2)

 

 

Ingredient (% ) ( 96 )
Edible whey 23.57 19.27
Response fines —- 7.86
Oat greats, steam rolled 21.93 —
Corn, ground 15.0 --
Fat-whey blend 10.89 8.32
Fish meal 10.0 9.0
Soybean meal 48% 5.0 —-
Animal plasma 4.5 4.5
Soy concentrate, extruded 3.3 -—
Calcium carbonate .64 29
Antibiotic‘ .5 .50
Oat flour .5 45.32
Dicalcium phosphate .05 .68
L-Iysine .01 .09
Flavor .4 .4
CS blend 2.75 2.75
Copper lysine complex .1 .1
Microingredients2 .86 .92
Calculated Nutrient Analysis

Crude protein 2.5 22.5
Fat 10.0 10.0
Fiber 1.5 .82
Calcium 1.0 1.0
Phosphorus .8 .8

 

'Antibiotic provided 100 g chlortetracycline, 100 g sulfathiazole and 50 g penicillin per ton of feed.

2llllicroingredients include swine vitamin and trace mineral premix, vitamin A 18.18 KlU/kg, vitamin
03 2.18 KlU/kg, vitamin E 49.99 lU/kg, vitamin K 21.99 mglkg, menadione 7.27 mglkg, ascorbic
acid 9.76 mglkg, biotin 0.25 mglkg, choline 234.20 mglkg, 3 mglkg, niacin 63.63 mglkg,
pantothenic acid 45.45 mglkg, pyridoxine 10 mglkg, riboflavin 10.91 mglkg. thiamine 10.01 mglkg,
vitamin B,2 39.99 mcglkg, iodine 1.58 mglkg, iron 250 mglkg, manganese 39.37 mglkg, selenium

0.3 mglkg, zinc 250 "IO/K9.

40

Table 2. Composition of Phase 2 (CI 14-33 in Exp. 1
and d 14-28 in Exp. 3)

 

 

Exp. 1 Exp. 3
Ingredient 1%) CA)
Corn, ground 50.05 49.83
Soybean meal, 48% 31.21 33.02
Standard wheat midds 6.74 5.18
White grease 4.0 3.67
Edible dried whey 2.74 2.74
Fish meal 1.5 1.50
Dicalcium phosphate 1.38 1.36
Calcium carbonate .73 .97
Antibiotic1 .5 .5
Salt .29 .31
L-lysine .1 .1
Copper lysine complex .05 .05
Flavor .2 .2
Microingredients’ .58 .57
Calculated Nutrient Analysis
Crude protein 21.18 21.5
Fat 6.3 6.0
Fiber 3.15 2.76
Calcium .8 .9
Phosphorus .7 .7

 

‘Antibiotic provided 100 g chlortetracycline, 100 g sulfathiazole and 50 g penicillin per ton of feed.

2Microingredients include swine vitamin and trace mineral premix, vitamin a 11.36 KlU/kg, vitamin
D3 1.36 KlU/kg, vitamin E 50 lU/kg, vitamin K 13.75 mglkg, menadione 4.54 mglkg, biotin 0.1
mglkg, folic acid 3 mglkg, pantothenic acid 28.41 mglkg, riboflavin 6.81 mglkg, Vitamin B,2 24.99

mglkg, iodine 1 mglkg, iron 160 mglkg, manganese 25 mglkg, selenium 0.3 mglkg, zinc 160
Ins/ka-

41

Table 3. Growth performance of weanling pigs fed supplemental zinc from
inorganic and organic forms (Exp. 1 and 2)

 

Supplemental Zn:

 

Zn as ZnO 0 0 500 0 0 500 3000
Zn as ZnAA 0 0 0 250 500 250 0
Zn as ZnMet 0 250 0 0 0 0 0 SEM
Exp.1
ADG, kg
(0-14 d) 269‘” 281'” .318‘ .301' 268'” .301' .236” .018
(14-33 d) .422 .456 .433 .427 .443 .415 .444 .024
(0-33 d) .357 .382 .382 .373 .368 .366 .356 .018
ADFI, kg
(0-14 d) .302“ .302“ .346‘ .320' .303“ .331 ' 274” .016
(14-33 d) .593“ .631 "’ .657' .587“ .61 1" .555” .612“ .030
(0-33 (1) .470 .491 .521 .472 .480 .460 .469 .021
G:F
(0-14 d)c .887" .932‘ .918' .940‘ .885“ .904“ .855b .020
(14-33 d) .712 .723 .658 .726 .726 .771 .724 .040
(0-33 d) .760 .778 .734 .789 .769 .805 .757 .028
Exp. 2
ADG, kg
(0-20 d) .220 .267 .291 .226 .026
ADFI, kg
(0-20 d) .265 .303 .342 .260 .026
G:F
(0-20 d) .831 .880 .853 .861 .038

 

I”’Means in the same row with uncommon superscripts differ (P < .05).

CQuadratic effect of ZnAA dietary treatments (P < .05).

42

Table 4. Growth performance of weanling pigs fed supplemental zinc and
inoculated with Escherichia coli (Exp. 3)

 

Inoculation: Sham E.coll Sham E. Coli Sham E. Coli
Supplemental Zn:
Zn as ZnO 0 0 0 0 3000 3000
Zn as ZnAA 0 0 250 250 0 0 SEM
ADG, kg
(0-14 d) .157 .164 212 .163 .165 .074 .046
(14-28 d) .493 .487 .451 .499 .531 .561 .055
(0-28 d) 265 267 299 .275 284 .240 .036
ADFI, kg
(0-14 d) .174 .173 214 .191 .197 .111 .035
(14-28 d) .607 .640 .587 .653 .628 .653 .080
(0-28 d) .374 .388 .386 .404 .396 .361 .046
G:F
(0-14 d) .911 .949 .952 .855 .719 .515 .174
(14-28 d) .820 .751 .772 .776 .892 .862 .076
(0-28 d) .718 .686 .776 .688 .749 .657 .086

 

43

Table 5. Plasma mineral concentrations of weanling pigs fed
supplemental zinc from inorganic and organic forms (Exp. 1 and 2)

   

 

Supplemental Zn:
Zn as ZnO 0 0 500 0 0 500 3000
Zn as ZnAA 0 0 0 250 500 250 0
Zn as ZnMet 0 250 0 0 0 0 0 SEM
Exp. 1
Plasma Zn. uglml
Day 0 .81 .87 .79 .81 .79 .87 .74 .03
Day 33' .64“ .73” .75" .72" .74" .80“ 1.66‘ .04
Plasma Cu, pglml
Day 0 1.73 1.62 1.53 1.68 1.69 1.78 1.70 .06
Day 33‘ 1.39"c 1.59‘ 1.31“ 1.50'” 1.48" 1.55‘ 1.46” .05
Plasma Fe, uglml
Day 0 2.44 2.14 2.71 2.35 3.09 2.45 1.88 .28
Day 33 1.65' 1.80' 1.91‘ 1.83‘I 2.05“ 2.46" 2.96” .34
Exp. 2
Plasma Zn, rig/ml
Day 0 .90 1.02 .81 .93 .1 1
Day 20 .92” .96” 1 .00” 3.03‘ .08’
Plasma Cu, pg/ml
Day 0 1 .63 1 .96 1 .42 1 .82 .12
Day20 1.13 126 1.13 1.10 .10'
Plasma Fe, pglml
Day 0 2.09 1.65 1.32 3.77 .82
Day 20 1.60 2.05 2.54 3.40 1.1

 

“-‘Means in the same row with uncommon superscripts differ (P < .05).

‘250 ZnAA dietary treatment effect (P < .05).

'500 ZnO dietary treatment effect (P < .05).

'data that was log transformed prior to analysis Indicated by logSEM but actual means are
presented.

Table 6. Tissue mineral concentrations Of weanling pigs fed
supplemental zinc from inorganic and organic forms (Exp. 1)

 

 

Supplemental Zn:

Zn as ZnO 0 0 500 0 0 500 3000

Zn as ZnAA 0 0 0 250 500 250 0

Zn as ZnMet 0 250 0 0 0 0 0 SEM
Liver, pglg of wet tissue

Zn” 52.8" 59.5"“ 62.5"“ 77.5‘I 67.8“I 65.1”“ 580.8' .07"
Cu 9.58 8.40 9.55 9.51 9.95 921 8.41 .12"
Fe 8.63“ 10.37“ 828" 10.72' 8.12"” 8.48'b 8.33" .13"
Kidney, pg/g of wet tissue

Zn 246" 24.6" 24.8" 23.7” 23.5" 24.3" 862' .05"
Cu 7.87” 7.88" 7.15" 8.33” 627“ 6.85” 42.1' .13"
Fe 19.9” 23.5” 21.3” 31 .8" 20.5" 19.9" 45.8' .12"
Metacarpal Bone, #919 of DM

Zn 767" 83.6” 81.6" 75.4" 81 .5" 90.4" 1522' 7.3
Cu .57 .53 .51 .54 .61 .64 .57 .08"
Fe 472" 61 .4'” 53.5“ 58.0"" 57.8“ 542'” 65.0‘ .1"

 

II'""‘"“Means in the same row with uncommon superscripts differ (P < .05).
’250 ZnAA dietary treatment effect (P < .05).
’250 ZnAA ‘ 500 ZnO dietary treatment effect (P < .05).
“Linear ZnAA dietary treatment effect (P < .05).

”Quadratic ZnAA dietary treatment effect (P < .05).
"data was transfonned prior to analysis but, actual means are presented and the logSEM.

45

Table 7. Tissue mineral concentrations of weanling pigs fed supplemental
zinc from inorganic and organic forms (Exp. 2)

 

SuemplpentaZn: * I A" i i

 

 

Zn as ZnO 0 0 0 3000
Zn as ZnAA 0 0 250 0
Zn as ZnMet 0 250 0 0 SEM
Liver, ugly of wet tissue
Zn 107.1" 122.3“ 155.2c 11662' .1"
Cu 432 30.9 45.0 19.4 11.7
Fe 135.0 1402 133.5 123.0 15.2
Kidney, pglg of wet tissue
Zn 31.7” 312" 34.4" 231 .8‘ .15"
Cu 18.8” 12.9" 13.8” 68.9' .19"
Fe 16.0 16.4 16.4 25.1 4.6
Longissimus Muscle, uglg of DM
Zn 50.0 111.3 71.1 68.5 31.1
Cu 23.4 95.1 31 .0 52.9 47.5
Fe 44.0 43.9 41 .5 43.3 2.9
Metacarpal Bone, #919 of DM
Zn 1026.3” 984.6" 969.3” 2805.2" .06"
Cu 3 .25 3.1 3.6 3.11 21"
Fe 274.1 222.1 1952 279.4 512

 

""'°'°Means in the same row with uncommon superscripts differ (P < .05).
"data was transformed prior to analysis but, actual means are presented and the logSEM.

Table 8. Hepatic mineral concentrations Of weanling pigs fed
supplemental zinc and inoculated with Escherichia coli (Exp. 3)

 

Inoculation: Sham E.coli Sham E. Coli Sham E. Coli
Supplemental Zn:
Zn as ZnO 0 0 0 0 3000 3000
Zn as ZnAA 0 0 250 250 0 0 SEM
Liver, rig/g of wet tissue
Zn 54.5“ 65.9‘ 53.1c 552c 611 .4' 458.9” 37.9
Cu 10.3” 10.8" 18.1' 12.3 10.5" 11.1 2.13
Fe 148.5” 141.9” 131.8" 131.5" 3052‘ 327.4" 25.3

 

“-‘Means in the same row with uncommon superscripts differ (P < .05).

47

Table 9. Urine and feces mineral concentrations Of weanling pigs fed

supplemental zinc from inorganic and organic forms (Exp. 2)

 

 

 

Supplemental Zn:

Zn as ZnO 0 0 0 3000

Zn as ZnAA 0 0 250 0

Zn as ZnMet 0 250 0 0 SEM

Urine, mgld
Zn .48° .86" 1 .10" 3.01 ' .14“
Cu 23‘ 26“ .32“ .35“ .14“
Fe .36”c .46“ .53‘I 28‘ .16‘

Feces, mgld
Zn 108.3c 210.0" 221.0" 1022.1 ' .07"I
Cu 47.3 52.0 47.1 48.9 3.7
Fe 128.8c 157'" 132.9“9 165.7‘ .07‘

 

”"Means in the same row with uncommom superscripts differ (P < .05).
"data was transformed prior to analysis but, actual means and logSEM are presented.

48

 

Cu Balance

 

 

 

 

 

 

Figure 3. Effect of Zn forms on Cu balance of nursery
pigs (d 16-20). Cu balance did not differ significantly
between dietary treatments ( P > .05 ).

50

 

 

 

Fe Balance

 

 

 

 

 

Figure 4. Effect of Zn forms on Fe balance of nursery
pigs (d 16-20). Fe balance did not differ significantly
between dietary treatments ( P > .05 ).

51

 

APPENDIX A

THE EFFECTS OF ZINC SUPPLEMENTATION ON METALLOTHIONEIN
AND GUT MORPHOLOGY OF PIGS INOCULATED
WITH ESCHERICHIA COLI

MATERIALS AND METHODS

Seventy-two crossbred pigs (Duroc x (Yorkshire x Landrace), non creep
fed, were weaned at an average age Of 16 d and average weight Of 4.86 kg and
allotted by sex and litter to a 28 d trial with a 3 x 2 factorial arrangement Of
treatments. Six separate temperature controlled rooms were used with three
pens per room and four pigs per pen with ad libitum access to feed and water.
Each dietary treatment was represented by three replicates Of E. coli inoculated
and sham inoculated pigs. The dietary treatments across the two health statuses
were as follows: basal (control), basal + 250 ppm Zn as ZnAA, and basal +
3000 ppm Zn as ZnO. Phase 1 (d 0-14) and phase 2 (d 14-28) basal diets were
the same as those in Exp. 1. All diets (Table 1 and Table 2) were pelleted and
met or exceeded NRC nutrient recommendations (NRC, 1988).

At weaning, three rooms Of pigs received an oral dose of 5 ml Of
enterotoxigenic Escherichia coli (serotype 0149:k88 + non motile) inoculum (1.3
x 10"° CPU/ml), in peptone saline. A sham of peptone saline was orally

administered to pigs in the remaining three rooms. On (I 5, pigs that had

52

53

previously received E. coli were given another 5 ml Of E. coli inoculum (3.2 x 10‘
1° CFUlml). On d 7, one pig per replicate group (n=18) was killed. The
remaining pigs (n=54) continued on test for 21 d. On d 28, pigs were killed by
injection Of penibarbitol at ml for every 4.5 kg. Immediately, tissue specimens for
histological study were collected from the duodenum and jejunum. One gram of
duodenal mucosa was scraped with a glass slide, weighed, combined with a
homogenate buffer with protease inhibitors at a 1:4 ratio. Sample was
homogenized with a Ultra-Turrax T25 homogenizer (lKA-Labortechnik,
Germany). Homogenate buffer contained 0.2 mMOIIL of PMSF, 0.6 mglL Of
leupeptin, 0.9 mglL of pepstatin A, 10 mMOIlL of Tris HCI, 154 mMol/L Of NaCl
and 0.2 gIL Of NaN,_ Samples were then frozen at -80°C for later determination
of metallothionein. A second duodenum sample (10-cm) was taken adjacent the
first segment. A mid jejunal sample was also Obtained and both the duodenal
and jejunal samples were fixed in 10% phosphate-buffered formalin. Histological
measurements were completed according to Coussement et al. (1982) and
Moxley et al. (1989). Samples were stained with hematoxylin and eosin. The
length Of the villi (from the tip of the villus to villus/crypt junction) and depth of
crypt (junction of villus/crypt to base Of crypt), villuszcrypt ratio, brunner’s gland,
circular muscle, longitudinal muscle, and total thickness were measured with an
ocular micrometer on a minimum Of seven different places per small intestinal

segment.

54

Liver samples were frozen at -80°C. Metallothionein (MT) concentrations
in the intestine and liver were determined by the non-radioactive silver binding
assay according to Lee et al. (1991) with an assay volume of 1 ml. Liver
samples were combined with glycine buffer in a 1:4 ratio and homogenized.
Samples were boiled for 2 min at 100°C and then centrifuged (microcentrifuge,
Fischer Scientific, Pittsburg, PA) for 2 min to separate supemant fraction.
Intestinal and liver homogenates were combined separately with glycine buffer at
a pH of 8 and .3 ml of silver to reach a volume Of 1 ml. Samples were mixed and
incubated for 10 min to saturate the metal binding sites of MT. The unbound
metals (including excess silver) were removed by adding .1 ml Of hemolysate
from rat red blood cells followed by boiling in water for 2 min. Samples were
centrifuged to separate the supemant fraction and silver concentration was
determined with flame atomic absorption spectrophotometery (Smith-Hieftje
4000, Therrno Jarrell Ash Corporation, Franklin, MA). The amount Of silver in
the supemant fraction was assumed to proportional to the amount Of MT
present. MT was expressed in micrograms Of MT per gram Of wet tissue.

The hard from which pigs were Obtained were determined to be exposed
to the TGE (transmissible gastroenteritis virus) prior to weaning Of the pigs in
this study. No pigs in this study died, however vomiting in some pens did occur
for one to two days.

Statistical Analysis. Data for this experiment was analyzed using SAS

software for GLM procedure and least squares of means by treatment (SAS

55

manual, 1994). The mean differences between the treatments were detected by
comparison of least square means. Differences were considered significant at
P < .05. Individual pig was the experimental unit and treatment and room were

part of the model statement.

RESULTS AND DISCUSSION

The effects of Zn supplementation on MT concentrations in the intestine
and liver Of pigs inoculated with sham or E. coli are given in Table 10. Intestinal
and liver MT followed the same pattern in that pigs fed the ZnO diet in the sham
and E. coli inoculated pigs had greater ( P < .05 ) concentrations of MT than pigs
fed the control or ZnAA supplemented diet. These results are similar to those
reported by Carlson et al. (1997). They concluded that pigs supplemented with
high concentrations Of ZnO stimulated the synthesis Of a greater amount of MT
than the control or organic supplemented diets. However, contrary to results
also reported by Carlson et al. (1997), the control fed pigs did not stimulate the
synthesis of a greater amount of MT in the intestine than organic supplemented
pigs. Concentrations of MT in control and ZnAA supplemented diets from sham
and E. coli inoculated pigs in the intestine and liver where different (P < .05).
Proliferation of E. Call in the intestines Of the pigs in this study was minimal as
determined by morphological evaluation. The diets the pigs were consuming

contained an antibiotic (CPS 250) which may have affected the ability Of the

56

E. coli to proliferate. Mild signs Of scouring were seen only after the second

dose Of E. Coli and lasted for two days. Vomiting was exhibited and as stated
previously, TGE may have been prevalent in some rooms. Differences in gut
morphology are given in Table 11. Minimal differences were seen and do not

appear to reflect the dietary treatment.

Table 10. Concentration Of metallothionein (MT) in the intestine and liver Of

weanling pigs fed supplemental zinc and inoculated with

Escherichia coli (Exp. 3)

 

Inoculation: Sham E.coli Sham E. Coli Sham E. Coli
Supplemental Zn:

Zn as ZnO 0 0 0 0 3000 3000

Zn as ZnAA 0 0 250 250 0 0 CV.
Intestinal MT, pglg

d 28 17.5" 31.5” 15.9” 24.8" 324.5“ 352.7‘ 70.3
Liver MT. #919

d 28 274.4" 528.4” 308.5“ 284.0” 2972' 2566' 56.5

 

"WMeans in the same row with uncommon superscripts differ (P < .05).

57

Table 11. Morphological measunnents of duodenal and jejunal segments of
intestine from weanling pigs fed supplemental zinc and inoculated with
Escherichia coli (Exp. 3)

 

 

Inoculation: Shamr E.coli Sham E. Coli Sham E. Coli

Supplemental Zn:

Zn as ZnO 0 0 0 0 3000 3000

Zn as ZnAA 0 0 250 250 0 0 CV.

Duodenum (microns)
vIIus length 671' 661'c 538” 5588‘c 524'” 593"c 18.1
crypt depth 292 301 302 316 312 360 22.6
villuszcrypt 2.38‘ 2.23” 1 .85“ 2.01 "’ 1 .76” 1 .73" 28.1
bmnner’s gland 120" 103'” 110‘" 111'" 140' 87" 34.0
circular muscle 105 88 101 100 1 07 77 33.8
longitudinal muscle 57'" 53" 55'" 53'" 60‘ 38" 39.4
total thickness 1355 1327 1236 1297 1298 1250 12.3

Jejunum (microns)
villus length 551 568 533 510 567 532 1 9.7
crypt depth 197° 236'” 238'" 226‘” 239’” 264' 20.1
villuszcrypt depth 287‘ 2.46” 225” 2.38"” 2.47"" 2.06" 24.8
circular muscle 82‘" 91'” 76" 104'” 77" 118' 43.5
longitudinal muscle 47'” 57‘ 37" 45"” 38‘” 48‘” 41.3
total thickness 978 1 1 03 1 003 1023 1028 1 087 1 3.1

 

”’"Illleans in the same row with uncommon superscripts differ (P < .05).

58

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