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Supplemental Chromium for Stressed Baby Pigs:
Effects on Performance, Zinc and Copper Retention
and on the Immunological response of Cellular
Components of Peripheral Blood

presented by

Victor Siberio Torres

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SUPPLEMENTAL CHROMIUM FOR STRESSED BABY PIGS:
EFFECTS ON PERFORMANCE, ZINC AND COPPER RETENTION
AND THE IMMUNOLOGIC RESPONSE OF CELLULAR
COMPONENTS OF PERIPHERAL BLOOD
By

Victor Siberio Torres

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

DOCTOR OF PHILOSOPHY

Department of Animal Science

1996

ABSTRACT
SUPPLEMENTAL CHROMIUM FOR STRESSED BABY PIGS:
EFFECTS ON PERFORMANCE, ZINC AND COPPER RETENTION
AND THE IMMUNOLOGIC RESPONSE OF CELLULAR
COMPONENTS OF PERIPHERAL BLOOD
By

Victor Siberio Torres

Two experiments were conducted to determine the effects of dietary supplementation of Cr
to weanling pigs. Performance, Cu and Zn retention, immunologic response and the Cr x Cu
interaction were evaluated. In the first experiment 12 pigs were used in a 15-day digestibility
trial A basal diet was supplemented with: 0 (T1), 0.50 (T2), or 1.0 (T3) ppm Cr. Feces and
urine were collected every 24h for 10d and duplicate samples were digested with nitric and
perchloric acids for Cu and Zn determination. Numerically pigs in T1 had a greater retention
of Cu than pigs in T2 and T3. Likewise, pigs on T3 had a greater retention of Zn compared
to T1 and T2. Both these differences were not significant (P>0.10). Pigs on T2 and T3 had
1 and 2d of negative Cu balance respectively. In contrast pigs in T1 had a positive Cu balance.
Dietary Cu needs of the stressed pig may not be met by 22 ppm. N 0 negative balances were
observed for Zn. In the second experiment 96 pigs were used in two, 35-day trials. Phase
1 and phase 2 basal diets were supplemented with either 0 ppm Cr and Cu (T1 basal), 0.5
ppm Cr, 0 ppm Cu ('12), 0.5 ppm Cr and 250 ppm Cu (T3), or 0 ppm Cr, 250 ppm Cu (T4).
Pigs were assigned to 3 blocks by weight in a 2 x 2 factorial arrangement of treatments and
housed in 0.61 m2 stainless steel pens (0.15 m2/pig) . The floor space provided to the pigs

ranged from 6 to 59% less than the recommendations during the course of the experiment.

Phase 1 diet was fed for 14d before switching to phase 2. Blood was collected d0 and
weekly thereafter to evaluate lymphocyte function, phagocytic activity (PA) and microbicidal
ability (MA) of the neutrophils. Lymphocytes and neutrophils were separated by Ficoll-
Hypaque density gradient technique. Aliquot of lymphocytes was incubated with the mitogen
ConA, PWM and PHA and [3H]thymidine to evaluate the blastogenic response. Opsonized
baker yeast and neutrophils were incubated in a shaking bath at 37°C for one hour to
evaluate the PA and MA. Although T2 had a greater numerical ADG, ADF and a lower F/G
than the other treatments, these differences were not significant (P>.10). There were no
interactions. No significant differences were found in PA, MA, or in the response of cells to
mitogen stimulations. However a reduction in the subjectively-determined incidence of
diarrhea was observed in both trials for pigs receiving the Cr diets. In summary, dietary Cr
supplementation as provided by high Cr yeast had no apparent effect on either performance,

on Cu or Zn retention, Cr and Cu interaction or the immune response of baby pigs.

Dedicated with love to
my first grade teacher, Rosaura Perez who taught me to love school
And to
the Isabela, P.R. Agricultural Extension Agent, 1036 E. Abreu who showed
me to love agriculture and was my model for my career
And to
my family especially Mame, Can, Diana, Lurdes and Daniel
but very special

to the grace of God.

iv

ACKNOWLEDGMENTS

The author is particularly indebted to many people who helped him to pass through
this dark, twisted and laborious but interesting and rewarding tunnel called graduate school,
and he wishes to express his sincere appreciation.

First, he wishes to express his deepest appreciation and thanks to Dr. Melvin T.
Yokoyama, his major faculty advisor, for accepting him as his graduate student and for his
patience and continuous assistance during his course of study, for his friendship, and for his
wise guidance and helpful suggestions throughout the preparation and editing of his
dissertation.

Thankful acknowledgment is also due to Dr. Gretchen Hill, Dr. Dale Rozeboom, Dr.
Robert Bull, and Dr. Maynard Hogberg, members of his graduate committee, for their advise
and help in reviewing this manuscript, and because their friendly interest in the author’s
graduate program was always a source of encouragement.

The author is deeply indebted to Dr. James E. Jay, Assistant Vice Provost, Office of
Diversity and Pluralism, College of Agriculture and Natural Resources for the graduate
research assistantship during the first two years of his career and for providing partial funding
for the research project. To Dr. Maynard Hogberg, Head Of the Department of Animal
Science, for the graduate research assistantship during the last two years and for lending him
his lecture notes in his first course at MSU.

Special thanks to the University of Puerto Rico, Mayaguez Campus, who granted four
years leave of absence to pursue his graduate studies at Michigan State University and to his

fellow professors for their encouragement and support.

The author is deeply indebted to Peggy Bull who taught him the protocol for the
immunological research and for allowing him to use the immunology laboratory facilities and
to Lorie Culham who taught him the protocol for cell harvesting and for her patience and
continuous help.

Appreciation also is acknowledged to Jane Link and Dr. Pao Ku in the non ruminant
nutrition laboratory for teaching him the mineral analysis protocol, allowing him to use the
laboratory, and for their assistance and suggestions.

Specials thanks to Dr. Sue Hengemuehle for her advise and friendship.

The author is also especially grateful to the swine crew and his fellow graduate
students for their help.

Finally, the author is indebted to his wife Carmen, and kids Diana, Lurdes and Daniel
for their ever-present encouragement, providing the moral and spiritual support.

VERY, VERY SPECIAL THANKS TO GOD.

TABLE OF CONTENTS

LIST OF TABLES .................................................. ix
INTRODUCTION ................................................ .. . . . 1
LITERATURE REVIEW .............................................. 6
Historical and General Characteristics ................................ 6
Chromium as an Essential Nutrient ................................... 8
Sources of Nutritional Chromium ................................... 16
Metabolism ................................................... 17
Digestion ............................................... 18

Absorption .............................................. 18

Transport ............................................... 21

Tissue Distribution ........................................ 22

Chromium at Cellular Level and Metabolic Conversion ............. 24

Excretion ............................................... 28

Toxicity and carcinogenicity of chromium compounds ................... 30
Toxicity ................................................ 30
Carcinogenicity ........................................... 32

Chromium in Food Producing Animals ............................... 34
MATERIALS AND METHOD ........................................ 38
Experiment One ................................................ 38
Animals, Feed, and Experimental Period. ....................... 38

Feeding the Animals and Sample Collection. ..................... 39

Analytical procedures ...................................... 42

Statistical Analysis ........................................ 43

Experiment Two ............................................... 44
Animals and experimental period ................................... 44

Blood Sample Collection ................................... 48
Immunological Test ....................................... 48

Mitogen Preparation and Storage ............................. 48

Yeast Cell Solution Preparation .............................. 49

Cell Isolation ............................................ 49

Peripheral Blood Lymphocytes ......................... 50

Neutrophilic Polymorphonuclear Leukocyte ................. 51

Blasto genesis Assay ....................................... 51

Phagocytic Activity and Microbicidal Ability ..................... 53

Statistical Analysis ........................................ 53

RESULTS ......................................................... 56
DISCUSSION ...................................................... 78
IMPLICATIONS .................................................... 89
LIST OF REFERENCES ............................................. 90
APPENDIX ....................................................... 113

LIST OF TABLES

Table 1. Ingredient composition of the basal diet a ........................... 40
Table 2. Mineral analysis of the high Cr Yeast and the Basal diet ............... 41
Table 3. Ingredient composition of the basal diet experiment 2. ................. 46
Table 4. Chromium and copper concentration in the experimental feed. ........... 47
Table 5. Weight of pigs (kg) during the experimental period, experiment one. ...... 57

Table 6. Copper consumption in baby pigs supplemented with dietary organic Cr. . . . 58

Table 7. Copper excretion in baby pigs supplemented with dietary organic Cr. ...... 59
Table 8. Copper retention in baby pigs supplemented with dietary organic Cr. ...... 60
Table 9. Zinc consumption in baby pigs supplemented with dietary organic Cr. ..... 63
Table 10. Zinc excretion in baby pigs supplemented with dietary organic Cr. ....... 64
Table 11. Zinc retention in baby pigs supplemented with dietary organic Cr. ........ 65
Table 12. Average daily gain in grams ..................................... 68
Table 13. Average daily feed intake in grams ............................... 69
Table 14. Average daily feed gain ratio. ................................... 70
Table 15. Stimulation index e of cells stimulated by concanavalin A .............. 73
Table 16. Stimulation index 3 of cells stimulated by phytohemagglutinin. ........... 74
Table 17. Stimulation index 6 of cells stimulated by pokeweed mitogen. ........... 75

Table 18 . Phagocytic activity of the neutrophil. ............................. 76
Table 19 . Microbicidal ability of the neutrophil. ............................. 77

Table 20. Proximal analysis of the basal diet compared to NRC recommendations . . 113

Table 21. Amino acid composition of the basal diet compared to NRC. .......... 114
Table 22. Vitamin composition of the basal diet compared to NRC. ............. 115
Table 23. Mineral composition of the basal diet compared to NRC. ............. 116
Table 24. [3H]Thymidine uptake by unstimulated cells (CPM)e ................. 117
Table 25. [3H]Thymidine uptake by concanavalin A stimulated cells (CPM) ........ 118

Table 26. [3H]Thymidine uptake by phytohemagglutinin stimulated cells (CPM)° . . . . 119
Table 27. [3H]Thymidine uptake by pokeweed stimulated cells (CPM)e ........... 120

Table 28. Total pigs weight. ........................................... 121

INTRODUCTION

An important goal of the swine industry is to increase the number of pigs weaned per
sow per year. To achieve this goal, many swine producers have decreased the weaning age.
On the other hand, the immaturity of the immune and digestive systems of young pigs are of
major concerns in pork production (Gatnau et a1., 1993). Passive antibody—mediated
immunity derived from colostral immunoglobulin are very low at this age (Speer et al. 1959,
Miller et al. 1962, Porter, 1976), and according to Haye and Komegay (1979) the decreased
physiological maturity from stress of weaning at an early age lowers antibody synthesis.

Early-weaned pigs usually suffer a significant setback at weaning (post-weaning lag)
because of a change to a dry diet and considerable stress (Fowler and Gill, 1989). Stress
represents the reaction of the body to stimuli that disturbs its normal physiological equilibrium
or homeostasis, Often with detrimental effects (Khansari et a1., 1990). A number of social and
environmental factors stress the animal and alter its resistance to disease. Excess heat or
cold, crowding, mixing, weaning, limit-feeding, shipping, noise and restraint are stressors that
are often associated with intensive animal production and have been shown to influence
immune function in various species (Kelley, 1985). There is ample evidence that both
physical and psychological distress can suppress immune function in animals leading to an

increased incidence Of infections disease (Roth, 1992).

2

Weaning is certainly a stressful event for domestic animals. Piglets are usually
separated from the sow, handled extensively, regrouped with unfamiliar pigs, and shifted from
a liquid to a solid diet. Weaning at 2,3 or 4 weeks of age has been shown to decrease the in
vivo and in vitro response of porcine lymphocytes to phytohemagglutinin (Blecha et a1.,
1983). This is considered to be a measure of the pig's ability to mount a cell-mediated
immune response. The stress of being removed from the dam is believed to raise the blood
concentration of cortisol which has an immune suppressive efiect at physiological (W estly and
Kelley, 1984) and at high concentration (Jefferies, 1991) by inhibiting the synthesis of several
cytokines and cell surface molecules required for immune function (Auphan et a1., 1995). If
an animal is irnmunosuppressed due to stress, such as a preexisting viral infection,
irnmunotoxicants or nutritional factors, the nonspecific defense mechanisms may not be
functioning optimally. In addition, the Specific immune response may be Slow to develop or
inadequate, which can result in clinical disease due to an infectious agent that would
otherwise be controlled by a non-impaired immune system (Roth, 1992).

Studies with humans and mice have indicated that various stressors due to infection,
strenuous exercise, or trauma may have marked effects on glucose metabolism and increased
losses of chromium (Cr) in urine (Borel et a1., 1984; Pekarek et a1., 1975 ). Chromium is a
natural constituent of living matter and an essential trace element for both plants and animals
(Anderson, 1981; Squibb and Snow, 1993). Early data suggests that the predominant
physiological role of Cr may be as an integral component of the Glucose Tolerance Factor
(GTF) which is necessary to potentiate the action of insulin (Anderson, 1981). Thus,

metabolic processes that are regulated by insulin including carbohydrate, lipid and protein

3

metabolism may be influenced by Cr (Anderson ,1981). In stressed cattle, the urinary
excretion of Zn and Cu may also increase (Orr et a1., 1990). When these minerals are
supplemented, the animals performance may improve (Nockels, 1990, 1995). Pull (1990) in
a review of effects of trace minerals on immunity concluded that deficiencies of Zn, Fe, Cu,
or Se results in a lowered resistance to disease.

In research with mice, Schrauzer et al. (1986) suggested that supplemental Cr may
prevent stress induced urinary losses of Cu, Zn, Mn and Fe. Research by Chang and Mowat
(1992) showed a marked increase in weight gain and feed efficiency of calves fed
supplemental organic Cr from a high-Cr yeast during the first 28 days after arrival at the
feedlot. No effect was noted on performance during the later growing period, but a marked
reduction in serum cortisol and an increase in total immunoglobulin were detected. In the
same study, Cr supplementation also reduced morbidity. Primary antibody response to human
red blood cell was higher for Cr treatments. Steele and Rosebrough (1981) found that
inorganic Cr (chromium chloride) increased the 21-day rate of gain in turkey poults. Using
chromium picolinate as the source of Cr supplementation in pigs from 30 to 100 kg of body
weight has been shown to increase the percentage of muscle by 7% and longissimus dorsi
muscle area by 18% (Page et a1., 1993). Schroeder et a1. (1965) also reported that
supplemental inorganic Cr improved growth rates of mice and rats. Mertz and Roginski
(1969) also found that Cr supplementation reduced mortality in stressed rats subjected to
additional stress of acute hemorrhage. Schroeder et a1. (1965) reported that Cr reduced
mortality from an epidemic of pneumonia with female rats. Studies in rats and monkeys

confirmed that Cr deficiency decreases glucose tolerance. In addition, severe Cr deficiency

4

leads to decreased growth, Shortened life Span, elevated blood cholesterol, aortic
atherosclerosis and decreases sperm count (Guthrie, 1982). A synthetic glucose tolerance
factor containing Cr potentiates insulin activity in pigs and this glucose tolerance factor is
biologically active in pigs (Steele et a1., 1977).

There is no recommendation for Cr in swine diets (NRC, 1988) and most swine diets
are composed of ingredients from plant origin which are usually low in Cr. Drinking water
in the United States also supplies only minute amounts of Cr in the diet (Durpur, 1962). So
it is possible that conventional diets for pigs may be moderately deficient in Cr. We therefore
decided to study the effect of supplemental organic Cr on the weanling pigs.

Our alternate hypothesis was that stressed baby pigs at weaning and during nursery
period supplemented with organic chromium in the diet will respond more positively to
immunological challenges, grow faster, and more efficiently and excrete less zinc and copper
compared to those receiving a non Cr supplemented diet. The general objective of this
research project was to demonstrate the effectiveness of organic chromium supplementation
(High Cr Yeast) to baby pigs during stress periods at weaning and in the nursery and to
evaluate if there was an interaction between Cr and Cu supplementation. The specific
Objectives of this research were:

1. TO determine the average daily gain, feed intake and feed efficiency of pigs
supplemented with 0, and 0.50 ppm of Cr in the diet, using two levels of Cu (25 and 250
ppm) without antibiotic supplementation.

2. To evaluate the immune response measured by the ability of lymphocytes to

proliferate when stimulated by mito gen.

5

3. To evaluate the ability of neutrophilic polymorphonuclear cell to phagocytize and
kill invading organisms.

4. To determine the effect of Cr on the excretion of zinc and copper.

LITERATURE REVIEW

Historical and General Characteristics

The name chromium came from the Greek word "Chromos", meaning color, and was
given to this metal due to the pronounced and varied colorations Of chromium compounds.
The red color of ruby gemstones and green color of emerald gemstones are due to small
amounts of chromium. This metal is a transition chemical element of Group VIB of the
periodic table (Cotton and Wilkinson, 1980; Greenwood and Eamshaw, 1986). It is a hard
steel metal that takes a high polish and is used in alloys to increase strength and corrosion
resistance. This element was discovered in 1797 by the French chemist Louis-Nicolas
Vauquelin and isolated as the metal a year later (Squibb and Snow, 1993).

Chromium is a relatively abundant element. It is the twenty first most abundant
element in the earth crust, but occurs in a much greater concentration in the earth core and
mantle. The free metal is never found in nature. In natural deposits, it is always combined
with other elements especially oxygen. Most ores consist of the mineral chromite which is
the most important mineral and the ideal formula is FeCrZO4 (Encyclopedia Britannica,
1995).

Chromium exist in a range of valence states from -2 to +6. The most common
oxidation states are +6, +3, and +2, although a few compounds of +5, +4 and +1 oxidation
states are known. In biologicals materials only Cr (11]) and Cr (VI) poses the stability to exist
for extended periods of time. Chromium (VI) can form various species with the most

important being chromate (CrO42’) and dichromate (Cr2077’). This two ions form the basis

7

for a series of industrially important salts. In addition chromates and dichromate are both
potent oxidizing agents that can be relatively toxic to living organisms. Chromium (H1) is the
most abundant form of the element in nature. It is also the most stable Species in acid solution
and the most stable oxidation state in biological materials. Trivalent chromium compounds
such as chloride, oxide and sulfate are generally insoluble in water, on the other hand
chromium chloride hexahydrate and the acetate and nitrate are readily soluble. It readily
forms octahedral complexes with oxygen, nitrogen and sulfur (Kumpulainen, 1988). In
biological fluids, chromium forms organic complexes with many ions such as oxalate, amino
acids, proteins, (Standeven and Werrerhahn, 1989; Kortenkamp et al., 1991), nucleic acids
(Cupo and Wetterhahw, 1985; Snow and Xu, 1991; Snow, 1994) and small molecules such
as glutathione (Anderson, 1987; Snow 1994). Chromium (V) species can be observed
experimentally and may play an important role in the toxicity of chromium, however this
Species does not form stable biological compounds. Chromium (IV) is highly reactive and has
not been observed directly in experimental systems, but exists as an intermediate in the
reduction of chromium (V) (Klein et al., 1992 ; DeFlora et al., 1990).

The mineral chromium is an important element for humans for several reasons. First,
Cr is used as a raw material by industry and is for example, a constituent of stainless steel.
Second, it may exist in toxic concentrations in the work place and industrial waste waters
(Kumpulainen, 1988). Third, Cr is considered to be an essential trace element for man,

animals (Anderson, 1981) and has beneficial effects in plants (Huffman and Allaway, 1973).

8

On a global basis, about 10 million tons of chromite ore are used annually. Of this,
approximately 76% is used for metallurgical purposes, 13 % for refractory uses and 11% for
chemical applications (Squibb, 1993). A small amount is used in medicine and research. One
of the primary uses of Cr in medicine and biomedical research is the utilization of radioactive
51Cr as a means of tracing specific cell populations (Kumpulainen, 1988). Because the
chromate anion is readily accumulated and retained by living cells, cells can be labeled with
51Cr in vivo and their half life or distribution within the body followed by gamma ray
detection or autoradiography. By labeling red blood cells, the total blood volume, cell
turnover, splenic function and gastric bleeding can be measured. White blood cells also have
been labeled to study the distribution and function of these cells in vivo. Laboratory
techniques have been developed that provide a means of measuring immune cells functions
in vitro by measuring the release of Cr from pre-labeled target cells (Squibb, 1993). During
the last few years, scientists have been studying Cr as a supplement for food producing
animals, mainly due to its possible potential for serving as an irnmunomodulator and irnproven

03 performance.

Chromium as an Essential Nutrient
An essential element as defined by Cotzias (1967) has the following characteristics:
1) it is present in all healthy tissues of all living beings 2) its concentration from one animal
to the next is fairly constant 3) its withdrawal from the body induces reproducible the same
structural and physiological abnormalities regardless Of the species studied 4) its addition

either prevents or reverses these abnormalities 5) the abnormalities induced by deficiency are

9

always accompanied by pertinent, specific biochemical changes 6) these biochemical changes
can be prevented or cured when the deficiency is prevented or cured. According to Mertz
(1969) an element is essential if its deficiency results in reproducible impairment of a function
from Optimal to suboptimal.

Chromium deficiency has been induced by feeding rations low in available Cr in
animals (Mertz and Schwarz, 1959; Doisy, 1963; Schroeder, 1966; Davidson and Parker,
1974; Seabom et al., 1994) and with deficient diets in humans (Anderson et al., 1991). The
discovery of Cr as an essential element goes back to 1959. In an experiment with rats,
Schwartz and Mertz (1959) reported that with a diet based on Torula yeast, the animals
presented an impaired intravenous glucose tolerance. When these researchers supplemented
the diet with small amounts of brewers yeast, the abnormality was corrected. They suggested
that to maintain normal glucose metabolism in animals fed Torula yeast, a dietary agent
present in brewers yeast was needed. They called this hypothetical dietary agent Glucose
Tolerance Factor (GTF). This potentiating effect of yeast on hypoglycemic animals was
reported 20 years before, but the phenomenon was attributed to the effects of a high
content of vitamins in yeast (Glaser and Halpem, 1939). The active ingredient responsible
for the metabolic effects of GTF was later identified as trivalent Cr ( Schwarz and Mertz,
1959).

The partial purification of GTF yielded a substance with a molecular weight between
300 and 500 dalton, which was water soluble and heat stable (Mertz and Roginski, 1971).
Toepfer et a1. (1977) further purified and analyzed the structure of GTF and described it as

a complex of two molecules of nicotinic acid, a tri-peptide and Cr(III) (Toepfer et al,.1977).

10

The predominant amino acids are glycine, cysteine and glutamic acid, the components of
glutathione (Mertz et al., 1978). Mixtures of synthetic compounds with a similar
composition proved to be nearly identical to the described physical and chemical
characteristics (T oepfer et al. 1977). These synthetic compounds were highly active in the
in vitro systems, and acutely improve glucose and blood lipid concentrations in genetically
obese diabetic mice in which simple Cr compounds such as chromium chloride were
ineffective (Tuman et al., 1978). Various groups of researchers agree with this description
of glucose tolerance factor (Mirsky et al., 1980; Yamamoto et al., 1989.) However, Haylock
et al. (1983) does not consider glucose tolerance factor fiom brewers yeast a Cr complex, and
he described it as a cobalt rather than a Cr complex. During the last decade an interesting and
controversial Cr compound has been isolated and characterized. This substance called low
molecular weight Cr ligand (LMWCr) was isolated from human urine, mouse and rabbit liver
(Y arnamoto et al., 1987), plasma, urine and feces (Y amamoto et al., 1981), dog liver ( Wada
et al., 1983) and bovine colostrum (Yamamoto et al., 1984). It has a molecular weight of
1500 daltons, an its composition is believed to be Cr, aspartic acid, glutamic acid, glycine and
cysteine, but not nicotinic acid (Vincent, 1993; Yamamoto et al., 1989) although some other
substance was present. Because the compound was seen initially after injection of hexavalent
Cr, they thought that the function of the compound was detoxification. Later research has
shown that the compounds exhibit some biological activity on glucose oxidation by isolated
fats cell (Y amamoto et al., 1989) similar to GTF. So they concluded that LMCr plays an
essential role in both the detoxification and nutrition according to physiological demands in

mammals.

1 1

Of all Cr compounds, only trivalent species exhrhits biological activity, and it appears
that the most effective form of Cr is as GTF. The main physiological action of Cr appear to
be related to all insulin dependent systems in which Cr deficiency should result in diminished
responsiveness to the hormone especially relative to carbohydrate metabolism. It also exerts
an effect on lipid and protein metabolism and on certain enzyme systems (Mertz, 1969; Giri
et a1., 1990). Although research had shown that this is the main action of Cr, additional
potential sites of action can not ruled out. The mechanisms by which this occurs is not well
known, however, and the identity of the GTF is still controversial. Chromium (III) by
participating in the complex GTF appears to facilitate or initiate the formation of the disulfide
linkages between the intra-chain disulfide of insulin and the mitochondrial membrane
sulfhydryl groups (Christian et a1., 1963; Mertz, 1967; 1993) SO it may act as a cofactor
with insulin, or it may act independently at the membrane level in the production of peripheral
insulin receptors (Anderson, 1987) . Vincent (1994) disagrees with this theory. Indeed he
postulates that GTF seems to play no role in glucose metabolism in normal cells and acts only
as a dietary agent providing a readily accessible source of Cr. He also said that GTF could
be deleterious to insulin action in insulin dependent cells if present in sufficient
concentrations. Vincent postulates that the active form of Cr in insulin dependent cells is the
LMWCr complex and has a post receptor role inside these cells instead of a receptor or cell
membrane role and that the role of glucose tolerance factor is as a nutrient supplying Cr to
the Cr deficiency species in the cell (Vincent, 1994). Evans (1989) also postulated a post

receptor action.

12

Regardless of where Cr has its effect, it appears that Cr is required for normal glucose
tolerance in animals and humans. After Schwartz and Mertz demonstrated that Cr was
necessary to maintain a normal glucose tolerance in rats, other researchers reported similar
findings. Glinsmann et al. (1966) reported an improved glucose tolerance by Cr
supplementation in human subjects. In patients receiving total parenteral nutrition, glucose
intolerance was improved after Cr supplementation (Brown et al., 1986; Freund etal., 1979).
Davidson and Blackwell (1968) reported that the impairment of glucose tolerance of
monkeys eating a Cr free diet was restored to normal by Cr supplementation. Anderson et
al. (1991) fed 17 middle-aged human volunteers a low Cr (<0.4 umol/d) diet and then 4
umol of Cr or placebo was given. He found no change in control subjects who entered the
study with a normal glucose tolerance test, but the glucose tolerance test of subjects with
impaired glucose tolerance deteriorated further during the placebo period and significantly
improved again during Cr supplementation.

Other signs of Cr deficiency include elevated serum cholesterol and triglyceride
concentrations, peripheral neuropathy and metabolic encephalopathy. Experimental animals
maintained on a Cr deficient diet had impaired growth, decreased survival, fasting
hyperglycemia and glycosuria, elevated serum cholesterol concentrations, and sclerotic
plaques in the aorta (Schroeder, 1969). Stress tolerance was also significantly lower in
animals with Cr deficiency than in Cr—supplemented animals (Merts and Roginsky, 1969).
Seabom (1994) also reported lower cortisol concentration in guinea pigs supplemented with

Cr compared with those fed a Cr deficient diet (<0.06 ug/g).

13

Jeejeebhoy et al., (1977) reported that in addition to glucose intolerance, human
subjects receiving a Cr deficient diet show serious metabolic abnormalities such as impaired
growth, peripheral neuropathy, negative nitrogen balance, and decreased respiratory quotient.
Lack of Cr has been reported to reduce growth in malnourished children (Gurson and Saner,
1973). The role played by Cr in these processes is also poorly understood (Squib, 1994 ).

According to Schroeder (1966) Cr deficiency represented a significant risk factor for
cardiovascular disease.

Chromium may also have an effect on lipid metabolism. Schroeder et al. (1962)
showed that Cr supplemented to rats receiving a low Cr diet caused a decrease in serum
cholesterol. He also reported that serum cholesterol and fasting serum glucose concentration
were depressed by brown sugar (high in Cr) or chromium (HI), but a diet with white sugar
(without Cr) caused a relative increase in serum cholesterol and glucose concentration
(Shroeder, 1969). In a study with human subjects with elevated serum cholesterol, Cr
supplementation resulted in a 6% reduction of total cholesterol compared to 6% increase in
the control group (Wang et al., 1989). Li and Stoecker (1986) reported a reduction in lipid
concentration in the liver in obese mice when supplemented with Cr in the diet. Doisy et al.
(1976) observed that in addition to the reduction in plasma glucose and insulin concentration,
some human subjects with impaired glucose tolerance also respond to GTF supplementation
with a significance reduction in fasting serum cholesterol. Other studies have shown that Cr
supplementation decreases serum triglycerides and total cholesterol and increases high density
lipoprotein cholesterol (Riales and Albrink, 1981; Mossop, 1983; Page et al. 1993).

Chromium also appeared to inhibit the development of aortic plaques. With a Cr deficient

14

diet, rats that died of natural causes had a higher incidence of aortic plaques. Schroeder et
al. (1970) found a lower aortic Cr concentration in people who died from arteriosclerotic
heart disease compared with people who had died from traffic accidents.

Insulin besides playing a role in carbohydrate and lipids metabolism also stimulate the
accumulation of amino acids in many tissues and enhances the incorporation of amino acids
into protein (Flelig and Begman, 1995). So if Cr is a cofactor for insulin, it also probably
plays a role in protein metabolism. Indeed, Mertz and Roginski (1969) reported a stimulation
in growth of rats when Cr was supplemented to rats eating a low Cr diet as compared with
un-supplemented control. They found that the increase in body weight was associated with
a proportional increase in tissue protein which was not due to water retention or fat deposit.

In an experiment with guinea pigs, Seabom (1994) reported larger liver, epididymal fat pad,
testes and kidney weights of Cr supplemented animals compared with a diet deficient in Cr
suggesting that Cr is involved in both protein synthesis and glucose incorporation into fatty
acids. However, the detrimental effect of a Cr deficiency on growth did not affect
hydroxyproline and creatine excretion, and they concluded that the increase in body weight
in Cr supplemented animals was associated more with increased body fat and water in
glycogen than with increased protein synthesis. Page et al. (1993) reported that growing
finishing pigs with Cr supplementation in the diet had an increase in longissirnus muscle area
that could be related to an increase in protein synthesis. They also found that Cr affected
nuclear protein and RNA synthesis. Wenk et a1 (1995) found similar results. However, there

were no statistical differences compared to the control.

15

In protein synthesis, Cr probably facilitates the insulin stimulated incorporation of
amino acids into protein (Hambridge, 1974). Roginski and Mertz (1969) investigated the
efl’ects of Cr in amino acid transport. They demonstrated in heart tissue that rats receiving
a low Cr-low protein diet have an impaired capacity to incorporate several amino acids into
protein. This incorporation was enhanced by Cr (III) supplementation. The amino acid
affected were at amino isobutyric acid, an amino acid analogue, glycine, serine and
methionine. When insulin was injected, the amino acid analog uptake by heart tissue of Cr
supplemented rats was greater as compared to unsupplemented rats.

Chromium also exerts an effect on several enzyme systems (Mertz, 1969). Horecker
et al. (1939) reported that Cr stimulated oxygen consumption in a succinic-cytochrome c
dehydrogenase system. Depending on the concentration Cr can also activate or inhibit DNA
and RNA polymerase (Snow, 1994)

Chromium will also inhibit certain enzyme reactions, such as those that involve
bacterial urease, and [3 glucosidase, when added to the media in excessive amounts. The
stimulations and depressing effects of Cr appears to be related to the concentration of Cr in
the medium (Mertz, 1969).

If Cr is accepted as an essential nutrient, not as a drug, its apparent effect, like that
of any other essential nutrient, depends on the nutritional status of the test subjects. It
improves an impaired function or restores it to normal, if that impairment developed because

of a Cr deficiency (Mertz, 1993).

16

Sources of Nutritional Chromium

Compared to other trace minerals, the information about Cr concentration in foods
and feed or the form in which it exists in these commodities is very limited. Also, many of
these results may be high for Cr due to contamination during sampling and sample
preparation, as well as to improper analysis ( Kumpulainen, 1988). Although, its seems that
Cr exists naturally at relatively low concentrations in plants and animals, mainly because Cr
(111) does not penetrate easily through cell membranes (Giri et al., 1990).

The best known source of Cr is brewers yeast. This is also the most characterized
form of Cr in feed and is discussed elsewhere in this thesis. Plants are also a source of Cr.
Both Cr (VI) and Cr ([11) are transported across root tips (Huffman and Allaway, 1973), and
both have been isolated from plant products (Lyon et al., 1969; Skeffington et al., 1973).
The form in which Cr is present in plants and its availability have been studied by Starich and
Blincoe (1983) and Huffman and Allaway (1973). They reported that both Cr (VI) and Cr
(III) are absorbed from the soil by plants. Most Cr (V I) is reduced to Cr (1H) and then is
assembled into a medium molecular weight complex different from the GTF present in yeast
This complex is highly stable in conditions that would be encountered in food and feed
preparation. While this complex has not been characterized, it may contain a mixture of the
oxidation states of Cr. Similar results have been reported by Schroeder et al. (1962 ) for oak
leaves and corn Oil. They showed that this form of Cr complex is more available than other
inorganic forms of Cr.

The Cr concentration of cereals, pulses, vegetables, fruits (fresh and dried) condiments

and spices, edible- flower and meats have been also determined (Guthrie, 197 5; Kumpulainen

17

and Koivistoinen, 1977; Giri et al., 1990; Wilplinger et al., 1995). The mean Cr value for the
analyzed cereals was 0.55 ppm, with wheat having the highest concentration with a value of
0.74 ppm (Giri et al., 1990). Previous reports found similar concentrations (Guthrie, 1975;
Carry and Olson, 1975; Kumpulainen and Koivistoinen, 1977; Kumpulainen, 1978) for whole
grains. Schroeder (1971) reported a reduction by more than 70% of Cr in refined white flour.
Also, white sugar retained only a small fraction of the original Cr content of raw sugar.
Between pulses, the highest Cr content was reported in broad bean with 0.50 ppm. Soya
beans, a common ingredient in animal feed preparations, have a concentration of 0.27 ppm.
Figs have 0.97 ppm of Cr, which is the richest form of Cr in fresh fruits. Dried fruits and
black pepper contain the highest Cr concentration varying between 1.20 and 1.50 ppm (Giri
et al., 1990). This value is lower that previous reports of 3.90 ppm (Guthrie, 1975). Most
other foods contain less than 0.10 ppm. For ready-to eat cured meats, a mean concentration
of 0.60 ppm has been reported (Kirkpatrick and Coffin, 1975). Most sea foods are poor

sources of Cr, whereas liver, kidney, spleen and beef contain relatively high amounts.

Metabolism

The metabolism of Cr is quite different from that of other trace elements because of
the strict dependence on the chemical form in which the element is present (Saner, 1980).
The majority of studies related to Cr metabolism involve the intestinal absorption,
transportation, distribution to the tissues, mode of action and excretion. There is very little

information about digestion. The rat had been used as the primary animal model.

18

Several factors can affect the metabolism of Cr. Some of them are valency, inorganic
vs. organic sources, health status and interaction with other minerals including Zn, Fe and Mn

ant other components of the food.

Digestion

Chromium in feed and food can be found as: Cr (III), Cr (VI) (Starich and Blincoe,
1983), GTF (Anderson and Mertz, 1977) low molecular weight Cr compound in milk
(Yamamoto et a1, 1988), and medium weight Cr complex (Starich and Blincoe, 1983). When
hexavalent Cr is exposed to gastric acid it is reduced to trivalent Cr (Donalson and Barreras,
1966). In the small intestine, due to the neutral pH, the majority of Cr compounds precipitate
and turn into an insoluble substance with characteristics different fron the original compound,
and later appear in the feces. According to Saner (1980) and Evans (1993) small amounts
can be converted to GTF, and this transformation process is age related. Starich and Blincoe
(1983) studied the stability of brewers yeast GTF and medium weight chromium complex in

the gastrointestinal tract They found that both are stable in the alimentary canal.

Absorption

It is not clear what mechanism is involved in the absorption of Cr. In vitro
investigations using rat intestine suggest that Cr (HI) may enter into the mucosa] cell by a
process of either simple or facilitated diffusion (Mertz and Roginski, 1971). Based on animal
studies, its appear that the small intestine is the site of absorption. Chen et al. (1973)

performed some in vivo and in vitro studies and indicated that Cr absorption occurs mainly

19

in the jejunum, followed by some absorption in the ileum and the duodenum. Several factors
have been shown to affect the absorption of Cr. One of these factors is the valence state of
ingested Cr. Inorganic Cr in its trivalent state has been shown to be less absorbable than
hexavalent Cr. Chromium (V1) is able to cross cell membranes using existing anion (P04 and
$04) transport systems, while Cr (HI) with its positive charge does not readily enter cells.
The greater toxicity of Cr (VI) versus Cr (III) in biological system is in large part due to this
difference in cellular uptake (Snow, 1994). In an experiment with rats, the absorption of
trivalent Cr was around 2-3 %, of an oral dose (Mertz et al., 1965) compared to 3-6% for
hexavalent Cr (Mackenzie et al., 1959). In an experiment with normal humans, Donaldson
and Barreras (1966) found that the absorption of Cr was 0.1 tol.2% and 0.2 to 4.4% when
administered as an oral dose for trivalent and hexavalent forms respectively. Similar results
have been reported by Witrner et al. (1989, 1991). Both Cr (VI) and Cr (III) bind to
components present in gastric juice. In addition, acidic gastric juice possess the capacity to
reduce Cr (VI) to Cr ( III). In vitro studies in which Cr was exposed to acidic gastric
secretions showed similar results. The importance of the interaction of Cr with gastric juice
on intestinal absorption has been demonstrated in experiments comparing intragastric versus
intraduodenal administration of Cr. Absorption of Cr (VI) increased 6 tol9 % when doses
were administered directly in the intestine, while on the other hand absorption of Cr (III)
remained basically the same. These researchers concluded that the acidic gastric juice
reduced hexavalent Cr to the trivalent state which is poorly absorbed (Donaldson and
Barreras, 1966; Doisy et al., 1968). These results were confirmed in an experiment with rats.

Thus, hexavalent Cr was found to be poorly absorbed when administered orally, however

20

when it was delivered directly to the intestine, the absorption was greater than trivalent Cr.
The form in which Cr is administered is another factor that can also affect its absorption.
Although inorganic Cr is poorly absorbed, complex molecules of organic Cr are absorbed in
larger quantities. Casey and Hambridge (1980) reported that the amount of absorbed
brewers yeast Cr glucose tolerance factor is 10 to 25% of an oral dose. Starich and Blincoe
(1983) studied the absorption of medium weight Cr complex present in plants and reported
a 30% uptake. Robles et al. (1981) reported similar results. Other anions present in feed may
also affect Cr absorption. In vivo and in vitro studies in rats demonstrated that oxalate
significantly increased and phytate significantly decreased intestinal Cr absorption (Chen et
al., 1973). Using everted jejunum segments, Mertz and Roginski (1971) found that complexes
of Cr with several amino acids were absorbed more readily than inorganic Cr. Substances
such as glutamate and penicillamine will increase Cr absorption (Chen, 1973).

The absorption of Cr can be affected by other nutrients in the gastrointestinal tract.
According to Mertz and Roginski (1971) some minerals such as iron, manganese, titanium
and calcium may depress the absorption of Cr. Chromium and Fe may share a single
gastrointestinal transport mechanism. In iron deficient animals, more Cr is absorbed from
00;, than in iron supplemented controls, because Fe depressed Cr binding to transferrin
(Hopkins and Schwarz, 1964). Transferrin forms a complex with Mn as well as Cr (Hurley
and Keen, 1987). Manganese deficiency in the guinea pig induces a diabetes mellitus-like
syndrome characterized by decreased utilization of glucose (Everson and Shrader, 1968).
This may reflect an interaction between Cr and Mn as a deficiency of Cr decreases glucose

tolerance (Mertz, 1967). Another interesting phenomenon is the apparent interaction between

21

Cr and Zn. Whole body contents of an oral dose of 51Cr were greater in Zn-deficient than
in Zn-supplemented rats (Hahn and Evans, 1975). In an experiment with rats it appears that
Cr and Zn share a common metabolic pathway in the intestine. Both Zn and Cr eluted in the
same low-molecular weight fraction when mucosa] supernatant extracts were separated by
gel filtration, suggesting that in the intestine a similar ligand binds both metals (Borel and
Anderson, 1984).

Health status, can also affect Cr absorption. The intestinal absorption of Cr(III) in
man is from 0.5 to 1.0% (Doisy et a1., 1971) but in insulin requiring diabetes during the first
24 hours after a single oral dose of Cr, absorption increase two to four times more chromium

compared to normal subjects (Doisy et al., 1971).

Transport

The transport of Cr appears to be different for the organic and inorganic forms of Cr
(Doisy et al., 1976). After inorganic Cr is absorbed from the gut, it is transported in the
blood, bound to the B globulin fraction of serum protein, especially to the protein transferrin
(Hopkins and Schwartz, 1964; Yamamoto et al., 1984). This is the fraction that also binds
iron, but only 30% is usually saturated with iron, so the remainder can provide transport for
Cr. The affinity of transferrin for Cr and Fe is very similar, so two metals can compete for
binding sites. Sargent et al. (1979) reported that transferrin has two binding sites, and the
affinity for iron of these binding sites varied. At a low Fe concentration, iron binds to site
A and Cr to site B, but at high Fe concentration, both metals, compete for binding mainly for

site B. When Cr is administered in high concentrations, it can also bind nonspecifically to

22

other plasma protein fractions (Hopkins and Schwarz, 1964) but with a higher affinity for
albumin (Tongeren and Major, 1966; Doisy et al., 1971; Yamamoto et a1., 1983) and partly
to low molecular weight Cr compound (Y amamoto et al., 1983). Hexavalent Cr can also
penetrates the red blood cell membrane and bind to the globulin fraction of hemoglobin
(Hopkins, 1965). In contrast, trivalent Cr cannot cross the erythrocyte membrane and do not
migrate into the red blood cells (Snow, 1993).

Organic Cr seems to be transported as GTF-bound Cr (Doisy, 1976). If this theory
is correct, then a mechanism of absorption for GTF as is, should be present, or a mechanism
to reassembly it after absorption from its components.

All forms of Cr have a rapid blood clearance rate, whereas various other organs retain
Cr much longer. These findings suggest that there is no equilibrium between tissue stores and
circulating Cr concentrations. and thus, concentrations of plasma Cr do not reflect the body

Cr state (Saner, 1980).

Tissue Distribution

Tissue distribution and elimination of absorbed Cr depends upon the valence state of
the Cr as it enters the blood stream and the reducing capacity of the serum. As stated above
Cr (III) binds to serum proteins such as transferrin and albumin, then is distributed primarily
to liver, kidney and spleen. Liver is the most important organ in which Cr is metabolized
(Yamamoto et al., 1989). Chromium (VI) rapidly crosses red blood cell membranes by the
sulfate transport system (Sugillama, 1992) and accumulates in the blood. Trapping of Cr (VI)

in red blood cells occurs through an intracellular reduction of Cr (V I) to Cr (V) and Cr (IV)

23

species and then to stable Cr (1H) (Snow, 1994). These reduction is performed mainly by
ascorbic acid, riboflavin, glutathione, and the flavoenzymes cytochrome P-450 reductase and
glutathione reductase (Seabom et al., 1994; Sugillama, 1994), with subsequent binding of the
Cr to hemoglobin. Species differences in Cr blood concentration occur and may be due, in
part to differences in ability of Cr to bind to hemoglobin molecule. The presently accepted
normal concentration of Cr in serum and plasma is 0.1 to 0.2 ng/ml (V ersieck and Cornelis,
1980; Versieck, 1985).

Chromium concentration in other tissues is 10 t0100 fold higher than in the blood
(Borel and Anderson, 1984). In humans and maybe also in animals, the concentration of Cr
varies with the geographic location. In general, in areas of the world with a high incidence
of maturity-onset diabetes and atherosclerosis, the tissue concentration is low (Borel and
Anderson, 1984). Factors associated with age also affect tissue Cr concentration in humans
and rats. Hopkins (1965) injected radioactive Cr into young, growing, and mature rats and
measured the radioactivity. He found that the spleen, kidney, testis, and epididymis retained
higher concentrations of Cr compared to heart, pancreas, lung and brain. Yamamoto et al.
(1988) using low molecular weight Cr found similar results. Young growing rats retained
greater amounts of Cr in the bones, whereas mature animals showed higher retention in the
spleen, kidney, testis and epididymis. In humans, Cr concentration in lung, aorta, heart and
spleen decreases considerably within the first few months of life, while the liver and kidney
retain neonatal concentrations until after ten years of age (Schroeder et al., 1962). The lungs
are the only tissue in which subsequent increase in Cr concentration is universally observed,

probably because of contamination in air (Borel and Anderson, 1984). Chromium

24

concentration in the hair also decreases with age. Infants have a concentration of 0.90 ppm
compared to 0.44 ppm for children of three years old. (Hambidge, 1972; Gurson et al., 1975).

The liver and the spleen of the infant which are the long term body pools of Cr (Lirn
and Kusubov, 1983; Yamamoto et a1., 1989) have been shown to contain 8 and 15 ng/g dry
weight respectively (V ouri and Kumpulainen, 1987). In adult human livers, Lievens et al.
(1977) and Bailey et al. (1983) reported a range of 2 tolO ng/g higher levels for both
organs. Other tissues contain less than 5 ng/g. The Cr concentration in human milk was
demonstrated to be approximately 0.4ng/ml (Kumpulainen et al., 1983). The accepted
concentration of Cr in the urine is 0.11ng/ml (Kumpulainen et al., 1983).

At the subcellular level, the nuclear fraction contained high amounts of Cr (Edwards
et al., 1961), specially the nucleoprotein fraction (W acker and Vallee, 1959; Okada et al.,
1989). When the source of Cr is as glucose tolerance factor, the highest concentration

appeared in the liver tissue, followed by uterus, kidney and bone (Mertz et al., 1971).

Chromium at Cellular Level and Metabolic Conversion

Although the most accepted theory on how Cr acts at the cellular level is the direct
interaction of Cr with insulin, the exact mechanism has not yet been elucidated. Protocols to
demonstrate the site or mode of Cr, have been performed by measuring the biological activity
of rat isolated adipose tissue. Biological activity refers to the ability of cells to stimulate
production of CO2 from glucose as a function of insulin concentration. In an experiment
using epididymal adipose tissue from Cr deficient and Cr supplemented rats in cell cultures,

Mertz et al. (1961) reported that the glucose removal rate in the Cr supplemented rats was

25

significantly higher than the low Cr group. These studies showed that epididymal adipose
tissue from Cr supplemented rats had a glucose uptake identical with that obtained from Cr
deficient rats in the absence of added insulin. However, a significant difference in glucose
uptake appeared with the addition of insulin to the incubation medium. The increase in
glucose uptake by insulin was much greater in the adipose tissue obtained from Cr
supplemented rats. The effects of Cr on glucose uptake takes place in the presence of small
amounts of insulin. Insulin alone was ineffective, and small amounts of Cr were required for
the demonstration of an insan effect. Similarly, the addition of Cr in vitro significantly
increased the effect of insulin on the incorporation of glucose carbon into fat and the
oxidation of glucose to C02. It appears that Cr is more effective when glucose is the
substrate, because when acetate, whose transport is insulin independent, was used the effect
was less pronounced (Mertz, 1965).

Much greater glycogen formation from glucose after insuhn administration was found
in Cr supplemented rats than in deficient controls (Roginski and Mertz, 1969). The Cr
supplemented rats also showed higher glycogen concentrations in their tissues than did low
Cr controls. These results demonstrated that Cr deficient rats were less responsive to insulin
than Cr supplemented rats (Roginski and Mertz, 1969). Farkas and Roberson (1965) and
Farkas (1967) investigated the action of Cr on glucose metabolism on the rat lens. In this
case, the action of Cr without insulin was ineffective but in combination was effective. Mertz
and Roginski (1963) studied the role of inorganic Cr on the transport mechanism of glucose
to the cell, and the possible interdependence of insulin and Cr. They used d-galactose, a

sugar that depends on insulin for transport, and it is not metabolized by peripheral tissue. In

 

 

 

 

26

this experiment, the transport of d-galactose in Cr deficient rats increased Slightly but when
they added Cr the uptake of d-galactose was greatly enhanced. They interpreted these results
to means that Cr plays an important role in sugar transport across the cell membrane acting
as a cofactor for the action of insulin. Saggerson et a1. (1976) reported that Cr in general
stimulates glucose uptake and lipogenesis by rat adipose tissue when incubated in vitro. They
considered that this insulin like effect was due to an alteration of plasma membrane structure
that would facilitate sugar transport, but would not mimic insulin through the insan
stimulated second messenger process. Steele and Rosebrough (1981) reported that dietary
inorganic Cr accelerates the rate of glucose uptake by poult liver incubated in vitro, but the
effect was not necessarily due to a synergism with insulin.

Glucose tolerance factor has qualitatively the same effect on glucose metabolism as
Cr, but the effect is greater than any inorganic Cr compound (Mertz, 1969). A significant
increase of glucose oxidation by the addition of GTF was also observed only in the presence
of insulin (Roginski et al., 1970; Evans et al., 1973). Using different forms of Cr, Evans and
Bowman (1992) demonstrated that both glucose and leucine uptake are greatest when skeletal
muscle is cultured in the presence of Cr picolinate compared to Cr chloride, Cr nicotinate and
a control. Significant uptake of glucose and the amino acid by cultured skeletal muscle cells
occurred only in the presence of insulin and uptake was greater when the cells had been pre-
cultured in a medium containing Cr picolinate. In cells with Cr picolinate, both the quantity
of insulin initially bound to receptor and insulin internalization rate were markedly elevated.

They conclude that Cr picolinate may affect the action of insulin through an effect on the rate

 

 

rm

31m

27

of insulin internalization, which by an unidentified mechanism regulates the synthesis and or
insertion of insulin receptors into the plasma membrane.
During the past few years, Yamamoto et a1(l983, 1987) and Okada et al. (1989) have
been studying the action of Cr at the intracellular level. They injected Cr intraperitonealy or
intravenouslyin rats and followed its metabolism. They found that Cr is incorporated into two
chemical species. The first was described as a high molecule weight protein which contained
5 to6 atoms of Cr per molecule and has a molecular weight of 70 KD. This protein is induced
in regenerating liver by the administration of Cr to partially hepatectomized rats. Chromium
in this form can bind to nucleolar chromatin resulting in significant stimulation of RNA
synthesis (Okada et al., 1989). These observations suggest a regulatory role of the protein-
bound Cr in nucleic acid synthesis. Products of high amount of Cr consisting of 70% RNA
and 30% protein was reported early in beef liver (W acker and Valle, 1959). The second
product has been described as a low molecular weight chromium protein. It has a molecular
weight of 1500 D and consists of Cr, aspartic acid, glutamic acid, glycine, cysteine and an
undetermined substance. This protein exists as Cr-free apo-low molecular weight compound
in various organs of many species and quickly binds invaded Cr to form stable Cr-bound low
molecular weight Cr (Yamamoto et al.,1983). When Cr reaches organs especially the liver
and kidney, it is incorporated into this protein. Yamamoto et al. (1983) measured the toxicity
of LMWCr and found a lower toxicity, higher rates of urinary excretion and renal clearance,
and lower rates of renal tubular reabsorption and retention in kidney and liver. Its appear that
this protein has a role in detoxification and also a physiological role in Cr metabolism. They

also measured the biological activity in vitro by measuring the effects of LMWCr from cow

28

colostrum on [U14C] glucose conversion to 14C02 and [33H] glucose incorporation into lipids
in the presence or absence of insulin They found no effect of M-LMW Cr when added to the
incubation medium alone. On the contrary, when insulin was added to the incubation mixture,
the glucose oxidation to about 10% more than the value produced by insulin alone at a Cr
content of 0.15 ng/ml and to about 20-25% at 1.5 and 15ng/ml. Milk low molecular weight
Cr also caused an increase in the rate of incorporation of glucose into lipids of 16% alone and

22% combined with insulin.

Excretion

Chromium absorbed from an oral dose appears to be excreted mainly by the kidneys
with small amounts lost in hair, perspiration, and bile (Collins, 1961; Doisy et al. 1971; Mertz
,1993). Collings et a1. (1961) injected intravenous radioactive Cr (III) and Cr (VI) into
anesthetized dogs and followed the excretion pattern. Four hours later they measured the
urinary excretion of Cr and found a concentration of 25% for trivalent and 9% for hexavalent
Cr. Biliary excretion was less than 0.5%. Four days later the excretion values in urine, bile
and feces were 50%, 0.5% and 3.7% for trivalent and 20%, 0.5% and 3.7% for hexavalent
Cr respectively. Urinary elimination also predominates for Cr (VI) compounds, however
biliary excretion may play a somewhat larger role, particularly following exposure via
inhalation (Squib and Snow, 1993). The exact mechanism of Cr metabolism by the kidney
is not known. The urinary excretion rate of Cr after an intravenous injection is quite rapid
during the first eight hours, which represents the rapid clearance of injected Cr from the blood

into the urine. Chromium excreted in the urine is derived from the dialyzable fraction of the

 

K1

dt

V3

C0

pr.
in 1

61f

29

serum Cr, which includes glucose tolerance factor. The daily urinary excretion of Cr is
approximately 3 to 50 rig/24 hours (Davidson and Secrest, 1972; Wolf et al., 1974).
Schroeder (1968) demonstrated that glucose loading caused an increased Cr excretion in the
urine during the first 2 hour after loading. Whole body elimination of a single intravenous
dose of Cr ([11) in rats occurs in 3 phases with half life values of 0.5, 5.9 and 83.4 days. The
valence can also affect Cr excretion. Studies in rats indicate that Cr (VI) compounds are
more rapidly excreted than Cr (1H). The half life for Cr (VI) was 22 days compared to 92
days for Cr (III) (Squib and Snow, 1993).

The elimination of Cr from body tissues has been determined in the rat following a
single subcutaneous dose of Cr (VI). Lung ,kidney, brain, heart and testes elimination curves
consisted of two phases, with terminal half life values of 20.9, 10.5, 9.6, 13.9 and 12.5 days,
respectively. The biologic half life of Cr in blood after reaching a peak at 5 day was 13.9
days. Liver tissue elimination of Cr occurred in three phases with half life values of 2.4 hour,
2.2 days , and 15.7 days (Squib, 1993). It appears that Cr is excreted in the form of LMW Cr
protein (Y amamoto et al.,l988; Okada et al., 1983). Some Cr is reabsorbed from the filtrate
in the renal tubuli. Estimates of renal tubular reabsorption of filtered Cr have ranged from
63% in dogs (Collins, 1961) to 80-97% in man (Davidson and Parker, 1974; Rabinowitz et
a1. 1980).

Stress caused by trauma, infection, surgery, intense heat or cold all elevate secretions
of hormones, and either directly or indirectly affect Cr excretion (Mertz and Roginski, 1969;

Nielsen, 1988). Urinary Cr excretion and plasma cortisol were increased in runners

30

(Anderson eta]..1982) and traumatized patients 42 hours following hospital admission (Borel
and Anderson 1984).

Chromium present in feces is mainly dietary non absorbable Cr but minimal amounts
of absorbed Cr may be excreted by the gastrointestinal tract. Hopkins (1965) injected an
intravenous dose of Cr to rats and 8 hours later sacrificed them to determine the Cr present
in their intestinal feces. He found that nearly 1% of the Cr present was from endogenous

origin. Starich and Blincoe (1983) and Borel and Anderson (1984) reported similar results.

Toxicity and carcinogenicity of chromium compounds
Toxicity

As with other elements, Cr has a role as an essential nutrient but can also be a potent
toxic and carcinogenic agent. Its relative toxicity is dependent on the concentration,
properties, the ability of the compound to enter the cell, the intracellular redox metabolism
of the compound and the reactivity of the compound or its metabolites (Squib, 1993). High
concentrations of Cr compounds have been found to be toxic in cell free systems, in bacteria
and non-mammals eukaryote, in mammalian cells in culture, in plants, animals and humans
(Costa et al., 1984; Leonard and Lauwerys, 1980). Very high doses of Cr may be lethal or
produce severe systemic toxicity. In humans, Cr poisoning is limited to accidental ingestion
of chromic acid or chromate (Borel and Anderson, 1984). According to Langard (1980)
damage to the kidney, liver, nervous system and blood are the major causes of death.
Chromium exposure in working areas can cause allergies and skin ulceration, perforation of

nasal septum, and bronchial asthma (Langard, 1980).

31

Of all the Cr valences, Cr (V1) is the most toxic. The acute toxicity is poorly
understood (W itrner et al., 1994). However, the mechanisms of toxicity of Cr (VI) in cells
is thought to involve the reduction of Cr (VI) to Cr (HI) by intracellular reducing agents such
as ascorbate, glutathione, and microsomal enzymes. Chromium ([11) then binds to nitrogen
and sulfhydryl groups and is known to react with amino acids, proteins and nucleic acids.
Thus Cr ([11) although the most stable form of Cr available in environment can bind to DNA
and produce effects on DNA replication in cell free systems but it does not readily enter into
cells and therefore is less toxic than Cr (VI) in vivo. Likewise the bioavailability of Cr
compounds may depend on the route of uptake. Soluble Cr (V1) is taken up by all types of
cells via cellular anion transport channels, thus these compounds are bioavailable and toxic
to bacteria and lower eukaryote as well as to mammalian cells in culture and in vivo.
Insoluble and sparingly soluble Cr compounds are not bioavailable or toxic unless they are
first solubilized. In contrast, many types of mammalian cells, including human lung cells, can
take up insoluble Cr compounds by phagocytosis into membrane bound vacuoles (Snow and
Squib, 1993). The inside of the vacuole is acidic, thus once the metal particles are taken up,
they are slowly dissolved and the metal ions are released inside the cell where they can reach
the DNA and other constituents. When tested, insoluble Cr compounds have often given a
positive genotoxic response in mammalian cells, both in vivo and in vitro. Organic Cr

compound has a very low toxicity compared with inorganic (Saner, 1980).

3 2
Carcinogenicity

Metal compounds, as a class, are among the best documented of all human
carcinogens, but their mechanism are not fully understood (Snow, 1994). Chromium has
been the most extensively investigated metal with respect to genetic and related effects
(DeFlora, 1989). The carcinogenicity of Cr compounds also appears to result from the attack
on DNA by Cr ([11) formed in situ by reduction of Cr (VI) (Witmer et al., 1994). In cell free
systems, but not in whole cells, Cr (H1) can interact directly with DNA to produce DNA cross
links, strand breaks and other conformational changes (Bianchi et al., 1983). Chromium (VD
does not directly interact with DNA except at very high concentrations unless incubation
occurs in the presence of either biological or chemical reducing agents. However, within a
cell or in the presence of glutathione, hydrogen peroxide or other cellular reductants, the
reduced Cr(V) or Cr (HI) can also interact with DNA and proteins to form stable Cr (III)
complexes. Once formed, these complexes are Slow to exchange and intracellular Cr (III)
complexes are poorly repaired. Chromium ([11) compounds also alter the kinetics and fidelity
of DNA replication (Snow, 1994).

In bacterial and non-mammalian systems Cr (III) compounds are not as biologically
active as Cr (VI) in whole systems. Chromium induces DNA damage as measured by DNA
strand breaks, SOS induction ( an inducible response to DNA damage in bacterial cells) and
differential microbicide of cells that are deficient in DNA repair mechanisms. Chromium (V 1)
also induces various types of DNA mutations in bacteria and higher organisms as frame shifts

and base pair substitutions (Snow, 1993).

33

In mammals and mammalian cells, soluble and insoluble Cr salts are toxic in vivo and
to mammalian cells in culture. All forms of Cr induce some degree of chromosomal damage,
DNA damage or mutagenesis. Both Cr (VI) and Cr (III) compounds bind to DNA in vivo
(Cupo and Wetterhahw, 1985) but only Cr (VI) compounds induce DNA strand breaks,
DNA-DNA cross links (Cupo and Wetterhahw, 1985; Sugiyama, 1986) and chromosomal
aberration (Newbold et al., 1979). Human fibroblast cells are especially sensitive to
mutagenesis by all forms of Cr (Squib and Snow, 1993). All forms of Cr also induce
genotoxicity in vivo and both insoluble and weakly soluble Cr including Cr oxide have been
found to be carcinogenic to rodents. Chromium (VI) compounds induce tumors in rodents
at the site of injection. Most Cr (VI) compounds, regardless of solubility induce
morphological transformation in cultured rodent cells (DiPaolo et al., 1979; Bianchi et
al.,1983). Higher concentration of Cr (111) may be needed to achieve the same biological
effectiveness as Cr (V I). Soluble and insoluble Cr (11) and Cr (III) compounds are usually
negative in in vivo carcinogenicity assays. Soluble Cr (VI) compounds are taken up rapidly
by cells via the sulfate transport system and reduced intracellularly to Cr (III) which is
considered to be an ultimate intracellular carcinogen. The reduction process itself may also
contribute to the carcinogenicity of chromate (Norseth, 1986). Chromium (11]) ions cannot
permeate the cell membrane but insoluble Cr (1H) particles may be taken up by phagocytosis
and then dissolve intra cellularly (Elias etal,.1986).

There are conflicting reports concerning the mutagenicity of Cr (1H) compounds.
Chromium (III) compounds were found nonmutagenic in a number of studies (IARC, 1980.,

Bianchi et al., 1983, Sugiyarna et al., 1986. , Levis and Majome, 1981, and Levy and

34

Venitt, 1986), but were mutagenic in V79 cells (Elias et al., 1986.) and also increased the
frequency of sister chromatid exchanges in CHO3 cells (Venier et al.,1985). The toxicity and
carcinogenicity of organic Cr compound has been less study, however its been assumed that
these compounds have a very low toxic effect. Yamamoto et al. (1989) studied the toxigenic

effect of MMLWCr and concluded that it is a very nontoxic agent.

Chromium in Food Producing Animals

Chromium as an essential micromineral for mammals is mainly associated with the
metabolism of carbohydrates, lipids and proteins, due to its believed effect on potentiating
insulin activity. Although a daily recommended allowance of 1 to 4 umol/day of Cr, based
in obligatory losses and bioavailability of Cr in food, is recommended for humans (Mertz,
1993), the exact requirement has not been established. For food producing animals there are
no recommendations from the National Research Council.

During the past few years, the importance of Cr as a supplement for food producing
animals has been studied. The effects of different concentrations and forms of dietary Cr
supplementation had been reported in dairy cattle (Burton et al., 1993; Kegley et al., 1995;
Chang and Mowat, 1992; Subiyatno et al., 1994; Wright et al., 1994), beef cattle ( Burton et
al., 1994; Moonsie—Shaeger, 1993; Mowat et al., 1993; Smith et al., 1994), swine (Evock et
a1, 1993; Lindeman et al., 1993, 1995; Boleman et al. 1995; Page et al., 1993; Wang et al.,
1995; Word et a1, 1995; Hanis et al., 1995; Chung et al. 1995; Wenk et al. 1995), poultry
(Anderson et a1 1989; Steele et al., 1981; Jensen et al., 1978; Steele and Rosebrough, 1981)

lambs (Kitchalong et a1., 1993, 1995; Forbes et al. 1995) and fish (Bureau et al., 1995).

35

Variables including performance, carcass merit, and reproduction have been evaluated, with
varying results.
It appears that Cr supplementation is more effective during periods of heavy stress.
Several experiment have been conducted to evaluate the effect of Cr supplementation in
stressed animals. In dairy cows, 0.5 ppm of supplemental Cr during late gestation and early
lactation, two severe stress periods, increased milk production during the first 16 weeks of
lactation by 11% in primiparous cows but had no effect in multiparous cows (Subityatrno et
a1, 1995). Others reported metabolic aspects to be improved by Cr supplementation such as
gluconeogenesis and normalization in glucose, insulin and glucagon kinetics (Chang and
Mowat, 1994). During the early lactation period, cows supplemented with Cr, also have a
higher humoral and cell mediated immune response compared to nonsupplemented animals
(Burton et al., 1993). In beef cattle. Chang et al. (1994) reported an improvement of 16%
on rate of daily gain and 19% on feed efficiency in feeder calves during the initial 28 days in
the feedlot. Moonsei-Shager and Mowat (1993) found similar results. However, in an earlier
study, Chang (1993) found no effect and other researchers have also reported no effect
(Lindell et al., 1994; Kegley et al. 1995). Another aspect that has been evaluated in feeder
calves is the effect of Cr on diseases and immune response. An improvement in humoral and
cell mediated immunity (Chang and Mowat, 1992; Kegley et al., 1995) and a decrease in
morbidity (Moonsie-Shaeger and Mowat, 1993; Lindell et al., 1994) have been reported. In
lambs, growth rate, feed intake and glucose kinetics were not affected by Cr picolinate
supplementation (Forbes et al. 1995). In swine, Page et a1. (1993) found that supplemental

Cr, in the form of Cr picolinate increased loin eye area and percent muscling and decreased

36

back fat in growing-finishing pigs. Chromium chloride had no effect Similar results have
been reported by Lindeman et al. (1993), Mooney and Cromwell (1993) and Chung et al.
(1995). Wenk et al. (1995) also reported an improvement on carcass characteristics and an
increase in average daily gain and decrease in feed gain ratio but only when the source of Cr
was Cr chloride and found no significant effect with either high Cr yeast or Cr picolinate
[(C,H§N02)Cr]. Ward et a1 (1995) reported that in growing—finishing swine the differences
between treatments for metabolites, hormones and growth and carcass characteristics were
minimal. Wang et al. (1995) conducted two trials, using adequate and a low protein feed
supplemented with 200 ppb of Cr picolinate. He reported that feeding Cr picolinate to
growing finishing pigs did not affect growth and back fat thickness, but larger loin areas were
noted. Conflicting results in nitrogen utilization were reported. In one trial, nitrogen
retention was increased but no effect was found in a second trial. Harris et al. (1995)
concluded that Cr picolinate had no effect on growth performance or carcass characteristics
in pigs. Lindemann et al. (1995) reported that total pigs born, live pigs born and pigs at 21
days were not affected by Cr supplementation, however in a previous experiment he found
that 200 ppb organic Cr fed through growing and gestation in gilts increased number of pigs
born alive and 21 day litter weight (Lindeman, 1994 ). In poultry Cr (Ill) increased the rate
of conversion of glucose to acetyl CoA in turkey (Steele and Rosebrough, 1981) and five
ppm of Cr (III) improved the egg interior quality of laying hens (Jensen et al 197 8). In fish,
Ward et al. (1995) found no effect on final body weight and nitrogen and energy retention of
rainbow trout supplemented with Cr. He concluded that Cr had no effect on the parameters

of economic importance in this type of fish.

 

 

elbcti
CODCCUI
trike

Crsup;

animrlr

37

Why does Cr supplementation produce positive effects in some trials but have no
effect in other trials? The key points appear to be stress, Cr status of the animal and Cr
concentration of the feed. When the animal is marginally deficient, or the amount of Cr in
the feed is low, or a combination of both and the animal is under heavy stress, a response to
Cr supplementation may be possible. However, when none of these factors are present, the

animals will not respond to Cr supplementation

 

 

 

 

Experil

Animal

retenti. ;'
L'nix'crsr

12194-2:

 

 

Were use

castrated.

Wind.

MATERIALS AND METHOD

Experiment One
Animals, Feed, and Experimental Period.

This experiment was conducted to evaluate the effect of supplemental Cr on the
retention of copper and zinc in stressed baby pigs. This study was approved by the All-
University Committee on Animal Use and Care of Michigan State University (AUF number:
12/94-221-00).

Twelve weanling (21: 1 day), crossbred (S? Yorkshim-Landrace x 6‘ Hampshire)
castrated male pigs with an average weight of 6021065 kg BW, from four different litters
were used. At day-one of age, pigs were identified by ear notch, and at day-ten they were
castrated. The pigs were not vaccinated and no creep feed was provided during the lactation
period.

A whey-com-soybean meal basal starter diet (Table 1) was prepared and
supplemented with either 0.5 or 1 ppm of organic Cr. This basal diet was formulated to meet
the National Research Council (NRC, 1988) requirements for pigs of this age (Appendix).
The source of organic Cr was a high Cr yeast product (Cofactor III), kindly provided by
Alltech Co. (Nicholasville, KY) with a concentration of 1000 ppm of Cr. The actual
concentration of Cr was confirmed by inductively coupled plasma general mineral analysis
procedure (Faires, 1982) before the product was mixed with the basal diet (Table 2). The
basal diet did not contain any antibiotics or supplemental copper. Forty six or twenty three

grams of the high Cr yeast was mixed with 0.45 kilograms of the basal diet and then with 45

38

sun

rm

and

Fee

l9:(
Iran
laid
urin
ptri

Cage

39

kilograms of feed for 25 minutes in a feed mixer, to prepare a diet with either 1.0 or 0.50 ppm
supplemental Cr. The final Cr concentration of the diet by analysis was 1.54, 2.12, and 2.61
ppm for T1, T2, and T3 respectively.

At weaning, pigs were weighed and assigned to three treatments in a completely
randomized block design (Gill, 1978). Treatments consisted of 0 (basal, T1), 0.50 (T2), and
1 ppm (T3) of supplemental Cr. The experiment had a duration of 15 days; first five days
were used to adapt pigs to diet and environment. During this time the pigs were housed
individually in a 0.84 m x 0.43 m elevated stainless Steel with screened floor cages. Basal diet
and deionized water were provided ad libitum. The housing temperature was maintained
at 30°C. During day six, the animals were individually weighed and transferred to 0.61 x

0.79 m metabolic stainless steel cage for total collection of feces and urine.

Feeding the Animals and Sample Collection.

To avoid contamination of feces with feed, the pigs were removed three times daily
(9:00 AM, 3:00 PM, and 9:00 PM) from the metabolic cages for about 30 minutes and
transferred to the adaptive period cages to be fed (Y okoyama et al., 1982). A rubber pad was
laid on the floor and an aluminum pan was placed under each cage to collect any feces or
urine voided during the feeding period. By closely watching the pigs during the feeding
periods, no losses were incurred in feces and urine. Before moving the pigs back to their
cages, their snouts were cleaned with a wet (deionized water) paper towel. The meals at day

6 and day 16 were mixed with 50 mg of brilliant blue dye (F.D. & C Blue No. 1)

40

Table l. Ingredient composition of the basal diet 8.

 

 

 

 

 

 

 

 

INGREDIENTS ‘ ‘ " ‘ ‘ ’ =GRAMS/KILOGRAMJ%
CORN, DENT YELLOW 558.50
SOYBEAN MEAL DEHULL , 200.00
WHEY DRIED 150.00
FISH MEAL, MENHADEN 50.00
CALCIUM CARBONATE 10.00
MONO-DICAL PHOSPHATE V 15.00 ‘
MSU SWINE VIT-TM-PMX b i 7.50
SELENIUM E PREMIX ¢ 1 5.00
WHITE SALT 2.50
L-LYSINE V 1.50
TOTAL 1000.00
3 On as-fed basis

bProvided the following per kilogram of the complete diet:
Vitamin A, 4950 IU; Vitamin D3, 990 IU; Vitamin K, 3.3
mg; Riboflavin, 4.95 mg ; Pantothenic acid, 19.80 mg;
Niacin, 26.4 mg; Choline, 168 mg; Vitamin B12, 30 meg;
Manganese, 56.1 mg; Iron, 89 mg; Copper, 14.85 mg;
Iodine, .75 mg; Zinc 112 mg.

° Provided the following per kilogram of the complete diet:
Selenium, 0.3ppm, and Vitamin E,16.5 IU.

41

Table 2. Mineral analysis of the high Cr Yeast and the Basal diet.

 

 

 

 

 

 

MINERAL BASAL HIGH CR YEAST
___P_"_M 121—ET
ALUMINUM 90.90 20.50
ANTIMONY < 5.00 < 5.00
ARSENIC <2.50 < 2.50
BARIUM 2.79 1.86
BORON 11.60 < 5.00
CADMIUM <0.50 < 0.50
CALCIUM 5690.00 1410.00
CHROMIUM 1.54 988.00
COBALT <0. 50 < 0.50
COPPER 22.20 21.90
IRON 166.00 66.40
LEAD <2.50 < 2.50
MAGNESIUM 1690.00 1610.00
MANGANESE 66.10 11.40
MERCURY <10.00 < 10.00
MOLYBDENUM 1.59 1.38
PHOSPHORUS 6200.00 12200.00
POTASSIUM 8040.00 16600.00
SELENIUM <10.00 < 10.00
SODIUM 830.00 957.00
THALLIUM <12.50 < 12.50
g 111.0_0__= . , 57.20

 

42

(Lutwak and Burton, 1964; Marlett et a1, 1981). This dye colored the feces and allowed the
collection of feces voided during the experimental period by visual examination. Deionized
water was provided ad libitum. Feed consumption and refusals were recorded daily. A
sample of the feed offered and the total of the refusals were collected into plastic bags and
stored at -20°C until analyzed. Feces and urine were collected every 24 hours for ten days.
Feces were collected separately on a fine plastic screen under the expanded metal floor of the
stainless steel metabolism cages in which pigs were housed. They were collected into plastic
bags (Nasco Whirl-Pak) weighed and stored at -20°C until analyzed (Ilori et a1., 1984). Urine
was allowed to pass through the screen onto a stainless steel collecting tray and collected
untreated into plastic containers. The total urine volume was recorded and a sample of 100
ml was retained and Stored in plastic conical test tubes with screw caps (Sarsdet, W.

Germany) at -20°C until analyzed.

Analytical procedures

Feces were oven dried at 65°C in aluminum pans for three days, and then ground in
a waring blender (Model 1120, New Hartford, CT). Feed and refused feed were ground
through a l-mm screen using a Wiley mill. Duplicate samples of approximately one gram of
feed, 0.50 gram of feces and 17 grams of urine were weighed into 250 Phillip beaker. Twenty
ml of nitric acid (14M) and four ml of perchloric acid (10M) were added to the samples and
digested under a perchloric hood until approximately one ml of volume remained in the flask
(Analytical Methods Committee, 1960). The flask was removed from the hood and the

interior sides were washed with deionized water. When the flask was cool, deionized water

 

 

W115

Stat?

Ann
C(‘mg'j
anal):

3506M

43

was added to attain a net weight of 50 grams. When necessary, the samples were further
diluted. The concentration of Cu and Zn in the samples were determined by flame atomic
absorption spectrophotometry (model 11 951, Instrumentation Laboratory, Wilmington, MA).
The final concentration of minerals was calculated using the following formula:
PPM = [(D)(FWF-FW)(C)] + (SW)

PPM = concentration of mineral in part per million

D = final dilution

FWF = final weight of the flask

FW 2 initial weight of the flask

C = concentration of the mineral (ppm)

SW = sample weight.

Intake and fecal and urinary mineral excretion daily and for the experimental period

was calculated and adjusted as mg per kilogram of body weight (Kimmel et al.,l988; Lei et

al., 1993).

Statistical Analysis

The experimental design was a complete randomized block with four replications.
Animals were blocked by litter, and individual pig was the experimental unit. The data set
consisted of Cu and Zn values for each day and the average for the period. Data were
analyzed using the ANOVA procedure of Statistical Analysis System (SAS, 1988). The
general linear model used was:

4 4
Y5 = the individual value
a = grand mean
Bj = effect of block
Ti = effect of treatment
Eij = residual error
Differences was considered significant at the level of P< 0.05 and the Scheffe method was

used for mean comparison.

Experiment Two

In the second experiment two Similar trials were conducted (May-June and
September-October, 1995) to evaluate the effect of supplemental Cr on performance, cell
mediated in vitro immune response of lymphocytes and the phagocytic activity and
microbicidal ability of the neutrophilic polymorphonuclear leucocyte (NPL) of peripheral
blood. This study was approved by All-University Committee on Animal Use and Care of

Michigan State University (AUF number: 12/94—221-00).

Animals and experimental period

Forty-eight (24 castrated males and 24 females) weanling (21:1:1day), crossbred (9
Yorkshire-Iandrace x d‘ Hampshire) pigs with an average weight of 7.28: 0.40 kg, from nine -
different litters, were used in each trial. At one day of age, pigs were identified by ear notch

and the Standard management practices were performed, including castration at ten days of

45

age. The pigs were not vaccinated and no creep feed was provided during the lactation
period.

A whey-corn-soybean meal (phase 1) and a com-soybean meal (phase 2) basal diets
(Table 3) were prepared and supplemented with either, 0 ppm Cr and Cu (T1 basal), 0.5 ppm
Cr, 0 Cu (T2), 0.50 ppm Cr and 250 ppm Cu (T3), or 0 ppm Cr, 250 ppm Cu (T 4). The
basal diets were formulated to meet the National Research Council (NRC, 1988) requirements
for pigs of this age (Appendix). The source of organic Cr was high Cr yeast product
(Cofactor III) with a concentration of 1000 ppm of Cr, which was kindly provided by Alltech
CO. (Nicholasville, KY). The source of Cu was feed grade copper sulfate. The basal diet did
not contain any antibiotics or supplemental Cu. Thirty five grams of the high Cr yeast, or
forty two grams of copper sulfate, or thirty five grams of the high Cr yeast and forty two
grams of copper sulfate or forty two grams of copper sulfate were mixed with 0.45 kilograms
of the basal diet and then with 67.73 kilograms of feed for 25 minutes in a feed mixer to make
the diets with 0.5 ppm supplemental Cr, 0.50 ppm supplemental Cr and 250 ppm Cu and 250
ppm supplemental Cu. The final concentration of Cr and Cu for T1, T2, T3, and T4 are
presented in Table 4.

At weaning pigs were weighed and randomly assigned to the treatments. Each
treatment was replicated three times. The animals were housed in 0.83m x 0.73m elevated
stainless steel screened floor pen, four animals per pen (two males and two females)
providing an average floor space of0. 15 m2/pig. The Pork Industry Handbook (Fritsche and
Muehling, 1987) recommends a floor space allowance of 0.16 to 0.23 m2 for pigs from 6.8

to 13.6 kg and 0.28 m2 to 0.37 m2 for pigs from 13.6 to 27.2 kg. The provided floor space

 

Table

. hm
CUR
Sm
DRll
F 1811
CALI
Mm
.\lSl‘
Slilll
\l'Hn

L-Ll’.

 

IllTr

Table 3. Ingredient composition of the basal diet experiment 2.

46

 

 

 

 

 

INGREDIENT G/KM PHASE 1 PHASE 2
CORN 558.50 658.50
SOYBEAN MEAL 200.00 300.00
DRIED WHEY 150.00
FISH MEAL 50.00
CALCIUM CARBONATE 10.00 10.00
MONO-DICAL PHOSPHATE 15.00 15.00
MSU SWINE VIT-TM PMX" 7.50 7.50
SELENIUM E PREMIXC 5.00 5.00
WHITE SALT 2.50 2.50
L-LYSINE 1.50 1.50

fl 1000.00 1000.00

3 On as-fed basis

 

bProvided the following per kilogram of the complete diet: Vitamin A, 4950
IIJ; Vitamin D3, 990 IU; Vitamin K, 3.3 mg; Riboflavin, 4.95 mg ;
Pantothenic acid, 19.80 mg; Niacin, 26.4 mg; Choline, 168 mg; Vitamin B12,
30 mcg ; Manganese, 56.1 mg; Iron, 89 mg; Copper, 14.85 mg; Iodine, .75
mg; Zinc 112 mg.

° Provided the following per kilogram of the complete diet: 0.3ppm Se and

16.5 IU Vitamin E.

47

Table 4. Chromium and copper concentration in the experimental feed.

 

 

 

T1 _ 1.58 23.00 1.70 26.20 1.40 18.02 1.60 20.30
T2 ‘ 2.16 28.05 v 2.20   19.08 2.05 23.00   2.12 A 18.04
T3 2.25 263.00 2.35 258 2.12 > 268 2.23 248
T4 1.45 265 1.35 270 1.38 , 258 1.60 ' 261 *

 

 

 

 

 

 

 

 

 

 

 

ranged from 6 to 59 % less than these recommendations over the course of the experiment.
This was the source of stress which followed the stress related to weaning. Experimental
housing temperature was maintained at 28 to 30°C for fourteen days then was gradually
reduced to 20°C. Each pen had two nipple waterer and a three-hole plastic feeder. Feed and
tap water were provided ad libitum. In the second experiment a dose of 200 ug of Cr in 5
ml of deionized water was administered orally by gavage to the pigs in T2 and T3 during the
first four days of the experiment. Pigs in T1 and T4 received 5 ml of deionized water. Daily
observation was performed to assess the health of the animals.

The experiment had a duration of 35 days. Phase one diet was fed for fourteen days
before switching to phase two. Animals were weighed weekly. Records of feed offered and
refusals were recorded weekly. Average daily gain, average daily feed consumption and feed

gain ratio were calculated by week and accumulative.

 

 

to
“i
['5

131'.

$sz

phfi

hnn

lfiu

C(mm
ofL.

48

Blood Sample Collection

Twelve pigs from three different litters were blocked by litter and assigned at random
to the treatments. These animals were bled early in the morning (7:00 AM) at day 0 and
weekly thereafter by vena cava venipuncture into 10 ml sterile evacuated tubes containing 143
USP units of sodium heparin (Vacutainer, Becton Dickson, Rutherford, N .J .) for cell
isolation. An additional blood sample was collected into 5 ml sterile tubes without
preservative for serum collection. The blood samples were held at room temperature for
subsequent immunoassay later the same day. Fletcher et a1. (1987) had shown, that

phytohemagglutinin assays lose significant activity by holding samples overnight.

Immunological Test
Mitogen Preparation and Storage

Stock solutions of mitogen were prepared and stored at -70°C. Two hundred and fifty
mg of concanavalin A (ConA), a broad T-cell mitogen, 100 mg of phytohemagglutinin
(PHA), a potent T-cell mitogen, 40 mg of pokeweed mitogen (PWM), a broad T and B-cell
mitogen and 10 mg of E. coli lipopolysaccharide (LPS) a potent B cell mitogen (Sigma
Chemical Co: St. Louis, M0) were dissolved separately into 100 ml of Rosewell Park
Memorial Institute (RPMI) 1640 culture media (Gibco Laboratories, Grand Island NY)
containing 6 ullml of fungizone and 0.32 22le of gentamicin sulfate in addition to 1 mmol
of L-glutamine, sodium bicarbonate and 10% of fetal bovine heat inactivated serum. The

mixture was sterile filtrated with a 0.22 um filter (Millipore Corp., Bedford, Ma) and stored

49

in one ml aliquot making a stock solution of 2.5 mg/ml, 1mg/ml, 0.4 mg/ml and 1 mg/ ml of
ConA, PHA, PWM and LPS respectively.

Before the study, various concentrations of the respective mitogens were incubated
with isolated pig lymphocytes to determine concentrations for optimum response. A final well
concentration of 20 pig/ml, 16 [lg/ml, 16 ug/ml and 10 ug/ml gave the maximal response for
ConA, PHA, PWM and LPS respectively. Mitogen solutions were thawed immediately
before use and working solutions of 275 ug/ml (0.3 cc stock solution of ConA + 2.43 cc
RPMI), 176 ug/ml (0.40 cc Stock solution of PHA + 1.87 cc RPMI), 176 lag/ml (1 cc stock
solution + 1.27 cc RPMI) for ConA, PHA, PWM and LPS were prepared. Unused portions

of both stock solutions and working solutions were discarded (Barta, 1984).

Yeast Cell Solution Preparation

Dried baker's yeast (Fleischmanns Yeast Inc., Oakland, Ca.) was used as target cells
to measure the neutrophilic phagocytic activity and microbicidal ability in this study . Fresh
yeast solution was prepared as needed. A half gram of dried yeast was dissolved in 200 ml
of 0.9% saline and stirred for 20 minutes on a magnetic mixer. The yeast cell number and
viability were determined, using a Neubauer Hemocytometer under a light microscope and

adjusted with 0.9% saline to 2 x 106 cell per m1 (Simpson et al., 1979)

Cell Isolation
The peripheral blood lymphocytes (PBL) and the neutrophilic polymorphonuclear

leukocytes (NPL) were separated from the blood by differential centrifugation (Boyum,

50

1968) using the Ficoll-Hypaque density gradient technique. Sterile procedure under a

Bioquest Biological Cabinet was used throughout the entire procedure.

Peripheral Blood Lymphocytes

Blood was diluted 1:2 in RPMI 1640 culture media in a 45 ml centrifuge test tubes
(Beckton Dickinson, Rutherford, NJ) and mixed very gentle by inversion. Seven ml of diluted
blood was layered carefully over 3 ml of cell separation media (Histopaque 1077, Sigma
Chemical Co: St. Louis, MO, 1.077 g/ml density gradient) in a 17 x 100 mm sterile plastic test
tube with cap (Falcon, Williams Ca.) using a ten ml sterile pipet and then centrifuged at 400
xg for 30 minutes with a table centrifuge (HN-S Centrifuge, International Equipment) at 18-
20°C. After centrifugation the lymphocyte-rich layer was transferred from the buffy interface
into another sterile 17x100 mm sterile plastic tube with a sterile Pasteur pipette, diluted in
10ml sterile EDTA phosphate buffered saline (BPS) solution (18g NaCl, 7.16g EDTA-Na3,
2.16g KH2P04, 972.68g deionized water, pH 7.4) and centrifuge at 275 xg for 6 min. The
supernatant was discarded and the cell pellet was examined for red blood cell (RBC)
contamination. When RBC was present the pellet was suspended in 10 ml of Tris ammonium
chloride (TAC)(7.5g NH4Cl, 1.0 g Tris-HCl and 991.5 g deionized water, pH 7.4) and
incubated at 37°C in a water bath for 12 min to lyse the RBC. After RBC decontamination,
the cell suspension was centrifuged at 150 xg for 8 min. The supernatant was discarded and
the cell pellet was washed with phosphate buffered saline (PBS, 8g NaCl, 0.2g KC], 1.15g
NaH2P04 and 990.45 g deionized water, pH 7.4) 2x; the first followed by a centrifugation

at 225xg for 6 min and the second followed by centrifugation at 275xg for 8 min. Washed

51

mononuclear cells were suspended in 2 m1 of RPMI,1640 media and counted in a Neubauer
hemocytometer under light microscope. The viability of the cells was assessed by trypan blue
exclusion (Sigma Chemical Co: St. Louis, MO). The cell suspension was adjusted to 2x10°

viable cell per ml (Hussain, 1981).

Neutr0philic Polymorphonuclear Leukocyte (NPL) Cell Isolation

The blood samples were diluted 1:1 with RPMI 1640 medium. For each sample 7 m1
of the blood medium mixture was layered into each of two 17x100 sterile plastic tubes with
caps containing 4 ml of cell-separating media and centrifuged at 18° to 20°C for 30 minutes
at a relative centrifugal force of 400xg. After centrifugation the plasma and cell separation
media layers were withdraw very carefully with a 10ml syringe with a long blunt needle. The
RBC-NPL granulocyte pellet was suspended in 10 m1 of TAC and incubated at 37°C in a
water bath for 15 min to lyse the RBC and then centrifuged at 300xg for 6 min. After
centrifugation the supernatant was carefully removed and discarded. The pellet was washed
with PBS buffer and centrifuged twice at 250xg for 10 minutes at 18° to 20°C. The RBC
lysis step was repeated until the cell pellet was clean and slight greenish in color. The NFL
cells were counted on a Neubauer Hemocytometer and the viability was assessed by trypan

blue exclusion. The cell count was adjusted to 2 x 10° cells/ml in RPMI 1640 media.

Blastogenesis Assay
A cell suspension of PBL (100 ill) from each pig containing 2x105 cells (Hussain et

al., 1981) was dispensed into 12 wells of a round-bottom 96-we11 micro titer cell culture plate

52

(Corning Glass Works, Corning, NJ). The cells in the first three wells of each row were
unstimulated. To stimulate lymphocyte proliferation in the other nine wells a volume of 10
pl of Concanavalin A (Con A) with a concentration of 330 pg/ml, phytohemagglutinin (PHA)
176 pg/ml and pokeweed mitogen (PWM) 176 pg/ml were dispensed to each of three
samples. This made a final concentration of 20, 16 and 16 pg/ml of ConA, PHA and PWM
respectively. The plates were then covered with Mylar sealers (Dynatech Laboratories Inc.
Chantilly, Va). Cultures were then incubated in a humidified incubator with 5% C02 at 37°
C for 4 days.

A total of l pCi of [3H] thymidine (specific activity 6.7 Ci/mmole, NEN Research
Products, Boston, Mass) in 20 pl volume was added to each well 18 to 22 hours before
termination of incubation period. After the incubation period the cell cultures were harvested
(Micro cell harvester, Straton Lier Norway) onto paper filter pads using TCA and deionized
water. Each well formed a disc in the filter pad and each disc was transferred to plastic
scintillation vials. A volume of 100 pl of soluene 350 (Packard Instrument Co., Downer’s
Grove, 111) was added to each vial to solubilize the cells. The vials were laid undisturbed
for 30 minutes and after that 4.5 ml of High Flash Point Cocktail Safety Solvent (Liquiflour,
New England Nuclear, Boston, MA) were added and the vials capped and labeled properly.

The incorporation of [3H] thymidine into newly synthesized DNA of stimulated and non
stimulated cells were determined with a liquid scintillation counter, (TM Analytic Inc., Elk
Grove Village, 111) three five minutes rounds per sample, and the results were recorded as
counts per minute (CPM). Stimulations indices (SI) were calculated as follows:

SI = RSC + RUC

53

SI = Stimulation index
RSC = average counts per minute in stimulated cells

RUC = average counts per minutes in unstimulated cells

Phagocytic Activity and Microbicidal Ability

To evaluate the ability of neutrophilic polymorphonuclear leukocyte (NPL) cells to
phagocytize and kill invading organisms the methods described by Simpson et al. (1979) was
used. Dried baker's yeast was used as target cells. Equals volume (200 pl) of yeast
suspension , NPL cell, autologous blood plasma, and RPMI 1640 were dispensed into tubes
with caps and incubated at 37°C in a shaking water bath for 60 min. After the incubation
period, 1 ml of 0.01% methylene blue was added to the mixture and was centrifuged at 400xg
for 10 min. The supernatant was discarded and the cell pellet re-suspended in 20p] of RPMI
1640 and observed under a light microscope with a Neubauer hemocytometer. The number
of PMN cells containing 2 or more yeast cells per 100 cells were counted to quantify the
phagocytic activity. The microbicidal ability of NPL cells was determined by counting the

number of NPL cells containing 2 or more dead yeast particles per 100 NPL cells.

Statistical Analysis

The experiment design was a randomized complete block with three repetition in a 2
x 2 factorial arrangement of treatments. Animals were blocked by weight and litter. In the
performance evaluation, the pen was the experimental unit and in the immunological

evaluation individual animal was the experimental unit. The data set for performance

54

consisted of average daily gain, average daily feed consumption and feed gain ratio values for
each week period and accumulative. For the immunological test the data set was stimulation
index for ConA, PHA, PWM and LPS and the values for phagocytic activity and microbicidal
ability. The day 0 values for SI and phagocytic activity and microbicidal ability were used as
covariate. Differences was considered significant at the level of P< 0.05. For mean
comparison the Scheffe test was used. The general linear model used for performance
evaluation was:

Yij=p+Bj+Ti+Eii

Yij = the individual value

,u = grand mean

Bj = effect of block

Ti = effect of treatment

Eii = residual error
The general linear model used for the immunological test evaluation was:

Yij =p+Bj+Ti+Cij+Eij

Yij = the individual value

p = grand mean

B J- = effect of block

Ti = effect of treatment

0 = effect of covariate

ll

Eij = residual error

55

All statistical analysis were performed in a Personal Computer using the General Linear

Model procedure of Statistical Analysis System (SAS, 1988).

RESULTS

Experiment one

This experiment was a digestibility trial to determine the effect of dietary chromium
supplementation on copper and zinc retention. It had a 15 day duration. The first five days
were the adaptive period, and the last ten days were the collection period. The first two days
were very stressful for the pigs. Pigs were highly agitated, jumping all the time trying to
escape, and barely ate the feed provided. Indeed they also lost some weight. On average the
pigs lost about 122 g/d during the first 2 days. The data related to the animals weight during
the whole period is presented on Table 5. The initial average weight was 6.02 kg. At day
4 they totally recovered and started to gain weight again. At day 5 they were back to the
initial weight. Based in average daily gain pigs eating the Cr supplemented diet (T2, T3)
recovered faster than those on the unsupplemented diet. All four pigs receiving the
unsupplemented diet and three in the 0.50 ppm of Cr developed diarrhea by the d8. In

contrast, pigs receiving 1.00 ppm supplemental Cr did not develop any diarrhea.

Effects of Cr supplementation on Cu retention.
The effect of Cr supplementation on Cu consumption, excretion and retention are
presented on Tables 6, 7, and 8. On average the amount of Cu consumed expressed as mg/kg

BW increased as the number of days in experiment progressed.

56

57

Table 5. Weight(kg) and performance of pigs, experiment one.

 

   

‘ 0441“” , 644
',nays~ } _ _   , “569
i 0444 5-89 404 , w F
" 6.,52 ’ 549 ~ _ 6424 . 602
, Day 12 643 ,7-27 +; I 747.
L ’Day 16 4 .» v.25 " _ 8.51. f i = 1845

    

 

. ”WW“ . .. _.
-4DG"g/d -186» _;214  , 243
. .ADFI‘g/d L ,265 _, w, . 3°41 A323 ,
FIGj , , 1.42 V ; I . .  _ '1.41 133 ”

 

 

 

 

ll.

 

a Weaning day

b Beginning of experimental period

c Beginning of collection period

d Last day of collection

° Basal diet

f Basal diet with 0.50 ppm supplemental Cr
3 Basal diet with 1 ppm supplemental Cr

1‘ Average daily gain

iAverage daily feed intake

ered gain ratio

58

Table 6. Copper consumption in baby pigs supplemented with dietary organic Cr.

 

 

        
   

   

'Da “Total . m BW Total . ‘ m- I BW.’ .TOjt'al f 4 BW
Day 1 3.26 N 0.55 4.16 0.65 . 4.09 w M 0.68 ,
‘Day2 4.64   0.79 4.99,. ‘0.80~.‘5.3610.89 ~
V .Day3 4.98 0.83 6.03 , . 0.96 _ V 5.11 . H 0.87
__ Day4 7 w' Q 5.08 0.85 v j , 4.96 5 — 0.82 g _ 5.05 ’1 .0.84~
"Days . 4.86 0.81 » , 4.45 . V 0.75 . > 5.47, . 0.91 .,
Days 1i ' 5.11 ’ i 0.73 , 5.52 t 0.77 34.297j-o.ss~,
, Day7 5.81 0.83 6.09 0.84 . 6.71 _ 0.93
5 Days   6.29 - * 0.90 e $6.754 " .10-93. "[9 I 6-86‘  .°497"
Day9 6.59 0.92 » 6.69 0.92 , N 8.32 ,, 1.17 ,
bay 10 7.64 T — 0.93? ' 7 ‘8»12 0-99' = 871* «1.28
a 5.33 0.81 5.69 0.84 6.24 0.94

 

 

 

 

 

 

 

 

 

 

a Basal diet

° Basal diet with 0.50 ppm supplemental Cr

° Basal diet with 1 ppm supplemental Cr

The data is an average of 4 pigs for treatment

Total is expressed as mg of Cu

59

Table 7. Copper excretion in baby pigs supplemented with dietary organic Cr.

 

b

 

 

Da Total mgllég BW
Day 1 3.05 0.51
Day 2 3.83 0.63
Day 3 2.90 0.48
Day 4 3.07 0.51
Day 5 4.39 0.74
Day 6 4.64 0.68
Day 7 4.01 0.57
Day 8 4.40 0.62
Day 9 4.58 0.65
Day 10 4.90 0.57

>‘< 3.88 0.60
a Basal diet

b Basal diet with 0.50 ppm supplemental Cr
c Basal diet with 1 ppm supplemental Cr
The data is an average of 4 pigs for treatment

Total is expressed as mg of Cu

 

 

 

 

Total mg[l_(g BW
2.98 0.46
3.52 0.56
3.79 0.60
4.78 0.78
3.63 0.61
4.21 0.60
4.75 0.67
5. 16 0.71
5.22 0.72
6.36 1.19
4.65 0.69

 

Total

2.39
3.57
4.70
5.1 l
4.19
5.48
8.40
6.20
5.30
8.09

5.37

 

 

60

Table 8. Copper retention in baby pigs supplemented with dietary organic Cr.

 

 

 

a

 

 

 

b

mykg BW
0.20

0.24
0.36
0.04
0.14
0.17
0. 18
0.21
0.21
-0.20
0.15

 

 

Total

1.69
1.79
0.41
-0.06
1.28
0.82
- 1.69
0.66
3.02
3.55
1.17

c
gig; BW

0.29
0.28
0.09
-0.01
0.21

0.1 1
-0.26
0.10
0.42
0.10

0. 13

 

 

 

Day Total mgllgg BW Total
Day 1 0.21 0.04 1.18
Day 2 0.81 0.16 1.47
Day 3 2.08 0.35 2.24
Day 4 2.01 0.34 0.18
Day 5 0.46 0.06 0.82
Day 6 0.47 0.05 1.31
Day 7 1.80 0.26 1.34
Day 8 1.98 0.28 1.59
Day 9 2.01 0.27 1.47
Day 10 3.28 0.37 3.80

>7 1.45 0.2_2__ =l__12
a Basal diet

° Basal diet with 0.50 ppm supplemental Cr

° Basal diet with 1 ppm supplemental Cr

The data is an average of 4 pigs for treatment

Total is expressed as mg of Cu

61

In general, pigs receiving no supplemented Cr (T 1) had a lower Cu consumption compared
to those receiving the supplemented diets ('12, T3). Pigs receiving 1 ppm supplemented Cr
(T3) had the higher Cu consumption during the whole period followed by those consuming
0.5 ppm supplemented Cr. In general, Cu consumption in T3 was from 5 to 38 % higher
compared to T1 and from 2 to 29 % higher compared to T2 during the period. Pigs in T2 had
a Cu consumption from 0 to 18 % higher than Tl. Pigs in T3 consumed 16 and 12 % more
Cu compared to T1 and T2, respectively. Treatment 2 had a 4% higher consumption
compared to T1. Although T3 had a higher consumption than either T1 or T2 the differences
were not statistically significant (P>0.10). The excretion of Cu was variable from day to day.
However, on average pigs in T3 had also higher Cu excretion compared to T1 and T2. Pigs
in T3 excreted 35 and 17 % more Cu compared to T1 and T2 respectively. Pigs in T2 excrete
15 % more Cu than T1. There were no significant differences due to treatment except for d3
when T3 and T2 had a significantly higher excretion of Cu compared to T1 (P<0.05).
Copper retention showed the opposite results. Pigs on the unsupplemented diet had a higher
retention of Cu compared to pigs on supplemented Cr. On the ten day average pigs in T1 had
7 and 5 % higher Cu retention than pigs in T3 and T2 respectively. Treatment 3 had the
lower Cu retention. Pigs in T3 and T2 had two and one day negative Cu balance. In contrast
pigs in T1 had a positive copper balance. However differences were not statistically

significant (P>0.10).

62

Effects of Cr supplementation on Zn retention.

The effect of Cr supplementation on Zn consumption, excretion and retention are
presented on Tables 9, 10 and 11. In general the amount of Zn consumption expressed as
mg/kg BW increase as the number of day in experiment progress. In general pigs receiving
supplemental Cr (T2, T3) had a higher Zn consumption compared to those receiving the
unsupplemented diet (T1). Pigs receiving 1 ppm supplemented Cr (T3) had the higher Zn
consumption during the whole period followed by those consuming 0.5 ppm supplemented
Cr. In general Zn consumption in T3 was from 8 to 36 % higher compared to T1 and from
0 to 26 % compared to T2 along the period. Pigs in T2 had a Zn consumption from 2 to 42
% higher than T1. On average pigs in T3 consumed 24 and 10 % more Zn compared to T1
and T2 respectively. Treatment 2 had a 12% higher consumption compared to T1. Although
T3 had a higher consumption than either T1 or T2 there were no statistically significant
differences due to treatments (P>0. 10). The excretion of Zn was also variable from day to
day. However, on average pigs in T3 had higher Zn excretion compared to T1 and T2. Pigs
in T3 excreted 42 and 11 % more Cu compared to T1 and T2 respectively. Pigs in T2
excreted 27 % more Zn than T1. Although there was a big difference in excretion between
T3 and T1 it was not a treatment but a block effect Retention of Zn was also variable from
day to day. On average pigs in T3 had the higher retention of Zn, followed by T1 and T2.
On average pigs in T3 had 8 and 9 % higher Zn retention than pigs in T1 and T2 respectively.
Treatment 2 had the lower Zn retention. Treatment 1 had 1% more Zn retention than T2.

All treatments had a positive balance of Zn. The differences were not statistically significant

63

Table 9. Zinc consumption in baby pigs supplemented with dietary organic Cr.

 

 

 

 

 

 

 

 

 

 

 

 

a b . ”c
' ngkg BW Total mgglég'BW '

Day 1 25.32 4.29 38.70 6.08 34.43 5.71
Day 2 35.37 5.98 49.69 7.90 47.50 7.96
Day 3 38.63 6.47 47.44 7.69 45.29 7.68
Day 4 40.75 6.80 41.77 6.93 44.25 7.36
Day 5 38.29 6.35 38.41 6.47 47.20 7.86
Day 6 42.45 6.09 48.16 6.73 54.03 7.52
Day 7 46.97 6.65 53.18 7.36 57.37 7.93
Day 8 52.36 7.38 58.10 7.98 59.91 8.46
Day 9 55.04 7.66 58.55 8.06 71.43 10.04
Day 10 56.44 8.04 63.18 8.65 77.95 10.94

>‘< 43.16 =657 49.72 7.38 fl 8.15

a Basal diet

° Basal diet with 0.50 ppm supplemental Cr
° Basal diet with 1 ppm supplemental Cr
The data is an average of 4 pigs for treatment

Total is expressed as mg of Zn

64

Table 10. Zinc excretion in baby pigs supplemented with dietary organic Cr.

 

 

 

 

a b c
& Total mg/kg BW Total—— 111 BW Total In BW ~

Day 1 15.01 2.48 15.65 2.34 13.74 2.20
Day 2 20.49 3.37 17.58 2.78 22.54 3.82
Day 3 14.61 2.44 20.59 3.25 30.02 4.97
Day 4 16.93 2.79 26.57 4.36 30.57 5.08
Day 5 25.11 4.24 22.05 3.71 25.16 4.24
Day 6 24.91 3.57 22.45 3.18 27.95 3.92
Day 7 22.42 3.16 33.21 4.68 42.79 6.02
Day 8 22.96 3.24 32.56 4.52 32.02 4.47
Day 9 24.17 3.44 30.49 4.19 26.14 3.69
Day 10 18.08 2.58 48.30 6.85 42.70 6.01

>‘< 20.47 3.13 Q95 3.99 29.3=6 4.44

 

 

 

 

 

 

 

 

 

a Basal diet

b Basal diet with 0.50 ppm supplemental Cr

° Basal diet with 1 ppm supplemental Cr

The data is an average of 4 pigs for treatment

Total is expressed as mg of Zn

65

Table 11. Zinc retention in baby pigs supplemented with dietary organic Cr.

 

 

 

, Day Total
Day 1 10.31
Day 2 14.88
Day 3 24.01
Day 4 23.82
Day 5 13.17
Day 6 17.55
Day 7 24.56
Day 8 29.41
Day 9 30.87

Day 10 35.94
>'< =22.45
5‘ Basal diet

 

a

m

BW
1.80
2.61
4.04
4.01
2.11
2.51
3.49
4.14
4.23
5.46
3.44

 

Total

23.05
32.11
26.85
15.20
16.36
25.71
19.97
25.55
28.06
36.28
2_9.4l

b

mg/kg BW
3.74

5.12
4.43
2.57
2.76
3.56
2.68
3.45
3.87
1.80
3.40

 

 

Total

20.69
24.96
15.26
13.68
22.03
26.08
14.58
27.90
45.29
48.70

 

25.92

 

b Basal diet with 0.50 ppm supplemental Cr

c Basal diet with 1 ppm supplemental Cr

The data is an average of 4 pigs for treatment

Total is expressed as mg of Zn

 

BW '
3.50
4. 15
2.71
2.27
3.63
3.60
1.91
3.99
6.35
4.93

3.70

 

66

Experiment Two

This experiment was conducted to evaluate the effects of supplemental dietary
chromium, and chromium x copper interaction on performance and immune response of baby
pigs reared in a crowded nursery environment. Two trials using forty eight pigs per trial were
conducted. In the first trial chromium was supplied only in the diet In the second trial for
the first four days 200 pg of Cr (cofactor III) dissolved in 5 cc of deionized water or 5 cc of
deionized water was orally administered daily. Pigs in the control diet and with copper
supplementation developed diarrhea by day 5 but not the pigs supplemented Cr. The diarrhea
problem lasted a week longer in pigs with supplemented Cu compared with the control.

No sign of other diseases or death losses were experienced.

Performance

The main effects of Cr supplementation on average daily gain (ADG), average daily
feed intake (ADFI), and feed/gain ratio (FGR) are presented in Tables 11, 12, and 13
respectively. Initial and weekly weight is presented in Table 27, in the appendix. There
were no statistically significant effects for Cr or Cr x Cu interactions for any of the response

criteria (P<0.10); therefore main effects are discussed.

Average daily gain (ADG)
In trial 1 in the first week all treatments had similar ADG, however in the second trial
ADG was suppressed ill pigs receiving 200 pg of oral Cr but not in pigs without extra Cr or

Cr and Cu supplementation. Average daily gain progressively increased during the duration

67

of the experiment except for week two in trial 1 when pigs gained less than in the first week.
On average, for the 35 day period in trial 1, pigs receiving the Cr supplementation (T2) but
no Cu and Cr supplementation (T3) had the overall better ADG. These pigs gained 16, 13
and 8% more than T3, T1, and T4 respectively. In the second trial pigs in the control group
(Tl) had the better ADG but the differences compared to the other treatments were less
marked than in the first trial. Pigs in T1 gain 5, 3, and 2 % more than T2, T4 and T3
respectively.

Average daily feed intake (ADFI)

Average daily feed intake progressively increased during the course of the experiment. The
ADFI was also greater for pigs on T2 in trial 1 except for week 1 where T4 had a better
consumption. In the whole period these pigs consume 16% more feed than T3 and 13% more
than T1 and T4, respectively. However in trial 2 pigs in T3 consume more feed than T1, T2
and T4. Again the differences between treatment in this trial were less marked and were less
than 5 %.

Feed gain ratio (FGR)

The FGR in trial 1 was better for T2 in the first 14 days, however from 14 to 35 days T4 had
the best ratio. With the overall trial T4 had a better numerical FGR and there were no
numerical differences between T1, T2 and T3. In the second trial during the first week T2
had the worse FGR and T4 the best, but during the trial T2 recovered and finally had a better
FGR than T4 and T3. On average T1 had the best FGR followed by T2. Treatment 4 was

the worse. All differences were not statistically significant

68

Table 12. Average daily gain in grams

lgfibwmawl
g T1‘ T2” T3c T4(1 T1 T2 T3 T4
0-7 189 207 187 194 91 59 107 128
7-14 142 176 172 148 214 213 236 233
0.14 164 191 179 169 152 136 172 180
14-21 316 358 280 326 266 285 271 306
0-21 217 249 214 224 190 185 205 222
21-28 468 493 412 476 412 395 380 356
0-28 282 312 266 289 246 238 248 256
28-35 528 622 556 568 578 532 537 485
0.35 333 376 325 347 312 297 306 302

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation
d Basal diet with 0 ppm Cr and 250 ppm Cu supplementation
Values per treatment are the average of 12 animals

eOral dose of 200pg Cr

69

Table 13. Average daily feed intake in grams

IMMI
_D_r_iy T1“ T2” 13° T4(1 T1 T2 T3 T4
0-7 220 241 240 255 154 122 167 173
7-14 312 345 337 309 395 385 454 485
0-14 270 297 293 284 274 254 310 329
14-21 506 612 492 482 506 502 522 576
0-21 353 407 363 353 351 337 381 411
21-28 809 892 715 790 703 699 761 696
0-28 471 533 454 466 439 427 476 482
28-35 1052 1185 1030 1072 1051 1011 1002 954
0-35 591 667 573 591 562 544 581 577

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation
d Basal diet with 0 ppm Cr and 250 ppm Cu supplementation
Values per treatment are the average of 12 animals

eOral dose of 200pg Cr

70

Table 14. Average daily feed gain ratio.

—-
. —

 
     

f 0.7 1.20 1.17 1.31 N f 1.32 N 1.83 , 2.45 N 1.98 . f 1.45
57-14 ‘ 2.33 1.96 ; :T 2.00  , .: j 2.11 f 1.92; 7.1.81}; 4.94. 2.07)}
0.14 1.65 1.56 1.67 1.69 1.89 1.87 1.83 “‘1-34,
0.21 1.62 1.64 1.69 1.58 1.85 1.82 N 1.86 1.85
21.23 1 ~. 1.73 7 1.81 7 1.74 , 1.66 V 1.72 _ 930.290.91.985.
0-28 1.67 1.71 1.71 1.62 1.81 1.81 1.92 1.91
as 200 m , ass/w 9......
0.35 1.77 1.77 1.76 » 1.71 11.81 1.84 1.90 .. 1.93

 

 

 

 

 

 

 

 

 

 

l
1'

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation
d Basal diet with 0 ppm Cr and 250 ppm Cu supplementation
Values per treatment are the average of 12 animals

°Oral dose of 200pg Cr

71

Immunological respome

The immunological response of the pig was measured as the ability of lymphocytes
isolated from peripheral blood to proliferate when stimulated by mitogens and the phagocytic
activity and microbicidal ability of the neutrophils. The proliferative responses for
nonstimulated and concanavalin A, phytohemagglutinin, pokeweed mitogen, and
lipopolysaccharide stimulated lymphocytes are presented in Tables 24, 25, 26, and 27 of the
appendix. The stimulation index calculated as the ratio between mitogen stimulated and non
stimulated cells are presented on Tables 15,16, and 17. The stimulation index for day 0 was

used as a covariate.

ConA mitogen: Table 15 summarizes the effect of Cr or Cr x Cu effects on Con A mitogen
stimulation of lymphocytes. There were no effects of Cr or Cr x Cu interaction between
treatments except for day 28 in trial 1 and 2. In trial 1 the animals receiving 250 ppm of
supplemental Cu had a significantly (P<0.02) better response than pigs receiving Cr, Cr and
Cu or no supplement. In the same period there were no differences between T1, T2, and T3.
However in trial 2 in the same period pigs receiving Cr and Cu supplementation had a
significantly higher response (P<.04) than T1, T2 or T3. In both trial there was a tendency

for a higher response for pigs with 250 ppm of Cu supplementation alone.

PHA mitogen: The response of lymphocyte to phytohemagglutinin mitogen stimulation is
presented on table 16. There were no significant differences between treatment in both trials.

However there was a tendency for pigs in T2 and T4 to have a greater response.

72

PWM and LPS mitogen. The effect of Cr and Cr x Cu effects on lymphocytes proliferation
stimulated by PWM and LPS are presented on Table 16. In trial 1 the mitogen PWM, a “b”
cell mitogen was used. There were no effect between treatment except for day 21, where
pigs in T3 and T4 had a significantly (P<.04) greater response compared to the control group
(T1) or pigs receiving only Cr supplementation (T3). In the overall period animals with
copper supplementation had the highest response although the differences were not
significant In trial 2 LPS mitogen was used and there were no statistically significative

differences. The stimulation index were similar for all treatrrrents.

Phagocytic activity and rrricrobicidal ability of the polymorphonuclear cells.

The phagocytic and microbicidal activities of the polymorphonuclear cells isolated
from the peripheral blood are presented on Tables 17 and 18. The phagocytic ability of PMN
as measured by the number of neutrophil that phagocytize two or more yeast cells per 100
PMN was un affected by Cr or Cr )1 Cu supplementation in both trials along the experimental
period. However, animals in treatment 4 tended to have a higher activity. The microbicidal
activity as measured by the number PMN that had two or more dead yeast cell per 100 PMN
was also unaffected. In the first trial all treatment had similar values, however in the second
trial those pigs receiving 250 ppm supplemental chromium had a higher microbicidal ability

compared to the other treatments.

73

Table 15. Stimulation index e of cells stimulated by concanavalin A.

 

 

 

 

[Day I T1“. ., .sz ,+ T38 9144“,; 4T1, _ T2T3 T4

 

Dayo _ 0.92 _ ,1-29 , 0-98, , 122 , 1.24 143 H .144 425
am 1.03 1.55 “1.59 ? 1.90 , 1.49 y "1.4a". 1.42‘ :‘1,43 
Day 14 1.60 1.09 1.10 > 1.56 0.74 0.98 0.82 0.98
”2721 1.15 ‘ . "1915' 0.85 s . 1.00 ; [0.78 995- ~‘i'0-687" 11.105"
Day28 1.31 _ 1.18 1.05 1.87 0.94 1.22 1.53 1.24
Dayss: 11.54 1 1.69 ‘ ’ 1.70 . 1.79 30.897122: 999] } 1.5.3.
7‘ L33 1-33 1:25 . 1-62. «95' 1-13.1-.09 1-27

 

 

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation

° Basal diet with 0 ppm Cr and 250 ppm Cu supplementation

e Ratio between thymidine uptake value by stimulated and nonstimulated cells.
Values are the log 10 of count per minutes.

Values per treatment are the average of 12 animals

eOral dose of 200pg Cr

74

Table 16. Stimulation index e of cells stimulated by phytohemagglutinin.

 

Day   T1“. 12" T34 ‘,T4° T1 , T213 T4

 

Day0 1.12 1.68 1.80 1.64 _1.71 1.66 1.74 ,_ 1.72
Day 14 1.66 2.03 1.80 1.80 _ 1.55 1.47 1.34 1.32
Day 21 , L60 .   1.34 ; ‘ 1.81 «1.92 .145 1.38 1133146
Day28 2.09 1.89 2.01 2.11 . 1.71 1.62 11.48 1.52
Day35 1.81 1.86 » 1.69 1.82    ?1.42..1.39.' 1.18-"1.48~~
52 1.81 ’ 1.91 , 1.88 1.92 1.50 1.45 V 1.36 _ 1.44

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation

° Basal diet with 0 ppm Cr and 250 ppm Cu supplementation

e Ratio between thymidine uptake value by stimulated and nonstimulated cells.
Values are the log 10 of count per minutes.

Values per treatment are the average of 12 animals

°Oral dose of 200pg Cr

75

Table 17. Stimulation index e of cells stimulated by pokeweed mitogen.

 

 

szf' ‘ ."1‘3c T46"; *   ' '

 

VDay0 . 1’02 1.64 . 1.43 1.53 1.08 1.33 1.34 1.38
as 7 .140 1461 1482 ‘ L96 1-21’.“1-23 .4237 127'-
Day 14 1.58 1.50 1.66 . 1.85 1.18 V 1.16 V V 1.07 1.03
1...... i... i...   1.7.1.91 ‘1...) 11.11....
Day28 1.46 . 1.56 1.47 1.90 V 1.33 1.34 1'19 . 1.25
04.135. * $150 » ~ 1.77 -: .1-81 ‘ 4.96 «  _ 121" 1.2011424 :5129~
V >7 V . 1.49 V 1.58 1.71 . 1.92 q 1.20 1.23 1.17 1.20

 

 

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation

° Basal diet with 0 ppm Cr and 250 ppm Cu supplementation

° Ratio between thymidine uptake value by Stimulated and nonstimulated cells.
Values are the log 10 of count per minutes.

“Oral dose of 200pg Cr.

76

Table 18 . Phagocytic activity of the neutrophil.

 

 

 

 

 

 

 

 

 

 

 

 

 

fig =.___...____——————_- — #
Day0 ’ 62 60 60 59 48 51 43 59
lDay7 72 70 72 71 53 47   45 57
Day 14 70 68 72 70 59 f 56 V w 52 _ 61 i
Dayzr ' 68   69 71 69   ’ 70   63 65 75
Day 28 71 73 75 74 68 65 63 70 i
Day35 72 71   72 ’ 67 ‘. 75 , 70  68 ‘ ”73
x _ 71 70 72 70 65 60 60 67
l_l_=.__=.=________ 1

 

5‘ Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation
° Basal diet with 0 ppm Cr and 250 ppm Cu supplementation
Values per treatment are the average of 12 animals

°Oral dose of 200pg Cr.

77

Table 19 . Microbicidal ability of the neutrophil.

 

 

 

 

Da T1“ T2b T3c T4“ T1 T2 T3 T4
Day 0 32 22 29 28 25 19 21 28
Day 7 38 30 41 35 28 27 26 25

Day 14 31 40 28 38 26 22 23 36
Day 21 40 27 3s 33 41 27 31 41
Day 28 39 40 38 41 37 33 35 38
Day 35 43 35 41 36 45 35 37 39

a 38 34 37 37 35 29 35 36

 

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation
° Basal diet with 0 ppm Cr and 250 ppm Cu supplementation
Values per treatment are the average of 12 animals

°Oral dose of 200pg Cr.

DISCUSSION

Experiment one
Weaning is certainly a stressful event for domestic animals. Piglets are separated from
the sow, handled extensively, commingled with unfamiliar pigs and Shifted from a liquid to
a solid diet. In this study pigs were weaned at 21 days and housed individually in pens where
they do not see each other. This certainly increased the level of stress. They were highly
agitated, jumping all the time trying to escape. When animals are stressed the level of plasma
cortisol increases (Rampacek et al., 1984; Becker et al., 1985) and is correlated with
suppressed immune function in pigs (W estly and Kelley, 1984). Peripheral concentrations of
this adrenal cortical hormone is an acceptable indicator of stress in swine (Strot, 1981). In this
study the level of plasma cortisol was not measured, however it has been well documented
in pigs that weaning, frustration, penning, mixing and handling will cause a significantly
increase in plasma cortisol concentration (Arnone, 1980; Dantzer et al., 1980; Hemsworth
et al., 1981; Barnett et al., 1981; Parrot and Misson, 1989; Roth, 1992; Burbolla et al., 1995;
Ekkel et al., 1995). All these variables were present in this study. When animals are under
stress simultaneously with an increase in cortisol. there is an increase in glucose metabolism.
Cortisol acts antagonistically to insulin because it prevents entry of glucose into peripheral
tissue to save it for tissues of higher demand, like brain (Burton, 1995). This results in a
mobilization of Cr body stores. Once mobilized, Cr is irreversible lost in urine (Mertz, 1993).
So if the diet has an inadequate Cr concentration, a deficiency may occur. An adequate

balance of nutrients is very important since an excess or deficiency in one component may

78

79

influence the availability or requirements for another (Roth, 1992). Studies with humans and
mice have indicated that various stressors due to infection, strenuous exercise, or trauma may
have marked effects on glucose metabolism and increased loss of Cr in urine (Pekarek et al.,
1975;Bore1 et al., 1984). Adrenocorticosteroid hormones can also affect urinary excretion
of Cu. Patients with adrenocorticosteroid insufficiency exhibit significantly lower urinary
Cu output than control, and normal patients given adrenocorticotropic hormone decreased
their serum concentration and increase their urinary Cu excretion (Davis and Mertz, 1987).
Orr et al. (1990) reported that in stressed cattle the excretion of Zn and Cu increase. A direct
relationship between plasma cortisol and urinary excretion of Cu and Zn had been reported
in humans and cats (Henkin, 1974). In food producing animals when these minerals are
supplemented, the animals performance may improve (Nockels, 1990, 1995). In research
with mice, Schrauzer et a1 (1986) suggested that supplemental Cr may prevent stress induced
losses of several microminerals including Cu, Zn, manganese, and iron. The results of this
study did not support this theory in pigs. We found that a supplementation of 0.5 or 1 ppm
of Cr to a basal diet already containing 1.54 ppm of Cr did not reduce excretion or improve
the retention of either Cu or Zn. Indeed, animals without Cr supplementation, although they
consumed less Cu per kg of body weight had a greater numerical retention of Cu compared
to those animals receiving 0.5 or 1 ppm of supplemental Cr. There was a linear reduction in
Cu retention as the level of Cr increased. Besides, pigs in the basal diet had a positive Cu
balance during the study, compared to one and two days of negative Cu balance for those pigs

receiving 0.5 and 1 ppm of supplemental Cr respectively.

80

Okonkowo et al. (1979) using a casein-glucose purified diet for 45 day-pigs,
concluded that 6 ppm of Cu provides an appropriate base for setting Cu requirements for
minimal adequate values of : average daily gain 200 g; hemoglobin 1.5 g/dl; plasma
ceruloplasmin activity 0.04 AOD 540/minute; plasma Cu 100 mg/dl; Cu balance 2 mg/d. Hill
et al. (1983) also found that 5 ppm Cu was adequate for producing normal weight and blood
and tissue parameters of young pigs fed a casein-glucose diet
In this study the feed concentration by analysis of Cu and Zn was 22 and 176 ppm
respectively. The Cu retention was 1.45, 1.42 and 1.17 mg/day for T1, T2 and T3
respectively. These numbers represent only 27, 25 and 19 % of the copper consumption. The
results reported in this study are lower than those reported by Okonkowo et al. (1979). In
Okonkowo’s study, pigs receiving 6 ppm Cu diet had a retention of 2.87 mg/day representing
49% of consumption.

When animals are under stress there is a substantial increase in Cu and Zn
metabolism. There is a substantially increase of ceruloplasmin and metallothionein synthesis
designed to cope with oxidant challenge induced by stress, since both these proteins can act
as free radical scavengers (Bremmer and Beattie, 1995). Glucocorticoid, cytokines such as
the interleukins and tumor necrosis factors, and other stress factors can induce
metallothionein synthesis principally in the liver but also in other tissues (Bremmer, 1987).
Blalock et a1. (1988) found that at low Cu intake levels, Zn is a more potent stimulator of
metallothionein gene expression in the small intestine than at higher Cu intake levels. Perhaps
a combination of stress and a relatively high Zn concentration compared to Cu, caused an

increase in metallothionein synthesis in the intestine, binding and preventing more Cu

81

absorption. This could be the reason for the lower Cu retention in this study as compared to
Okonkowo’s findings. Its possible that for stressed baby pigs, receiving a corn soybean diet,
22 ppm of dietary Cu is not sufficient to meet requirement.

In this study Zn retention was not also affected by Cr supplementation. The Zn
retention per kilogram of body weight was 3.44. 3.40, and 3.70 mg for T1, T2 and T3
respectively. These number represents 52, 46 and 45% of consumption. These results are
in agreement with those reported by Forbes and Yohe (1960), Smith et al. (1960) and O'Dell
and Savage (1957), but not with Lei et al. (1993) who reported a negative retention of -46%
of consumption.

In summary, 0.50 or 1.00 ppm of dietary chromium supplementation to a diet
containing 1.54 ppm of chromium as provided from high chromium yeast had no apparent
effect on copper or zinc retention of stressed baby pigs. It is also possible that for stressed
baby pigs, receiving a corn soybean diet, 22 ppm of copper is not sufficient to meet
requirements.

Experiment two

Performance: Early weaned pigs usually suffer a significant setback at weaning
characterized by a growth depression, associated with low voluntary feed intake (post
weaning lag), primarily due to a change to a dry diet and the accompanying stress (Fowler and
Gill, 1989). Stress represents the reaction of the body to any stimuli, that disturbs its normal
physiological equilibrium or homeostasis, often with detrimental effects (Khansari et al.,
1990). In order to alleviate this post weaning lag several approaches have been tried

including the addition of antibiotics and spray dried porcine plasma to the post weaning diet.

82

According to Mowat (1994) antibiotics may reduce certain stress symptoms, and the
administration of antibiotics to chickens reared in a dirty environment decreased immunologic
stress by reducing plasma interleukin-l levels resulting in improved growth (Klasing and
Roura, 1991). Spray dried porcine plasma has been shown to stimulate high feed intake and
average daily gain in the first two weeks after weaning (Owen et al., 1995; Zimmermann,
1987; Gamau and Zimmermann, 1990) reducing the post weaning lag. However, there is
concern about the feeding of antibiotics to animals because of the potential hazard of
developing of antibiotic resistant pathogens, which may constitute a human health problem.
Also, spray dried porcine plasma is a very expensive product to feed for very long. So other
promising ways of reducing this post weaning lag should be investigated.

It has been demonstrated that Cr supplementation in beef cattle can increase weight
gain and prevent or reduce depression in dry matter intake and in some cases even increase
feed intake (Moonsie-Shaeger and Mowat, 1993; Wright et al., 1994). In another study a
decreased body temperature by 0.5°C and a lower serum cortisol level in growing steers was
reported when their diet was supplemented with dietary high Cr yeast. An increase in the 21
day rate of gain has also been reported in turkey poults when their diet was supplemented
with Cr chloride (Steeele and Rosebrough, 1981). In this study, 0.50 ppm of dietary Cr
supplementation did not appear to help baby pigs cope with post weaning lag. In the first trial
although pigs receiving the supplemental Cr diet had a greater numerical average daily gain
during the first 7 days and also for the duration of the trial this differences was not significant
compared to the nonsupplemented animals or with the combination of Cr and 250 ppm of Cu.

In the second trial when an additional 200pg of Cr was administered as an oral suspension

83

for four days average daily gain was drastically reduced. When the additional Cr
supplerrrentation was stopped the animals recovered. The results of this study is in agreement
with the results of van Heughen and Spears (1994) who found that, although Cr
supplementation increased ADG and ADFI in comparison to control pigs it was not effective
in alleviating a reduction in performance due to stress caused by lipopolysaccharide injection.

The expected ADG, ADFI and FIG ratio for pigs between 5-10 and 10-20 Kg
according to NRC (1988) is: ADG 250 g/d and 450 g/d; ADFI 460 g/d and 950 g/d and FIG
ratio 1.84 and 2.11 respectively. However, restricted or inadequate floor space allowance
will decrease daily gains and feed intake of all classes of pigs, but is more pronounced in
weanling than in growing-finishing pigs (Komegay and Notler, 1984; Petheric, 1983). In this
study the provided floor space ranged from 6 to 59% less than Pork Industry Handbook
recommendation (1987). The data of this study shows that Cr or Cu supplementation to
weanling pigs reared in a crowded nursery environment is ineffective in alleviating the
reduction in performance due to a reduced floor space allowance as assessed by ADG and
ADFI. Compared to the expected values of NRC these pigs had a 30% and 37% less ADG
and ADFI respectively. On the other hand the FIG ratio was 14% better than expected NRC
values. Our results are in agreement with Kornegay et al. (1993).

It was surprising that the animals consuming the diet with 250 ppm of Cu did not
exhibit a greater response in ADG and FIG ratio compared to those receiving only 26 ppm
of Cu. This lack of beneficial response to supplemental Cu does not agree with a majority of
reports in the literature as summarized by Bowland (1990) nor do the results agree with more

recent studies by Apgar et al. (1995), Zhou et al. (1994) and Dove (1995) in which positive

84

growth responses and feed gain ratio improvement were obtained when supplemental Cu was
added to the diet. However, not all experiments in which 125 to 250 ppm of supplemental
Cu was added to the diet resulted in positive results. Indeed some researchers have reported
no effect of Cu supplementation and some have reported a reduction in performance from
high levels of Cu supplementation (Wallace et al., 1968; Parris and McDonald, 1968;
Livingston and Livingstone, 1968; Gipp et al., 197 3; Bradley, 1983).

The exact mechanism by which Cu stimulates growth and feed efficiency in pigs is not
fully understood. Some researchers postulate that this effect is primarily due to its
bacteriostatic properties (which have microbicidal effects) in the intestinal tract, similar to
antibiotics (Hawbaker et al., 1961; Miller, 1991). On the other hand, responses to antibiotics
generally are minimal when tested in a clean environment (Cromwell, 1991). In this study
pigs were reared in a very clean nursery environment isolated from other pigs. So the lack
of a beneficial response in performance compared to other studies may be explained by the
cleaner conditions and a more controlled environment than the conventional nursery.

Hemoglobin and hematocrit of pigs in all treatments in this study were within normal
ranges found in young pigs. This would suggest that the addition of Cr did not have an effect
upon whole blood parameters.

Pigs receiving supplemental Cr seem to have a lower incidence of diarrhea compared
to the other treatments. It has been reported that Cr compounds inhibit the growth of
microorganisms by interaction between Cr and the microbial DNA and also by the inhibition
of DNA polymerase activity (Ogawa et al., 1989). The etiology of the diarrhea in this study

was not established, however if it was caused by a bacterial infection then maybe Cr

85

supplementation inhibited microbial growth in the intestines of those pigs. Perhaps this could
explain the lower incidence of diarrhea.

Immunological Response: Several social and environmental factors stress animals and alter
their resistance to disease. There is ample evidence that both physical and psychological
distress can suppress the immune function in animals leading to an increased incidence of
infectious disease (Roth, 1992). Its has been reported that crowding and restraint will stress
pigs sufficiently to decrease their immune responsiveness. In a 28 days trial, housing eight
pigs (11.5-18.0 kg) per group in pens with 0.13 m2 of floor space per pig significantly
reduced their phytohemagglutinin skin test response as compared with pigs given twice as
much space (Yen and Pond, 1987). When young pigs were restrained by placing them in a
13 X 33 cm expanded metal box for two hours per day over a three day period, they had
significantly elevated plasma cortisol concentration which correlated with a decrease in the
size of their thymus gland and a reduction in the phytohemagglutinin skin test response
(Westly and Kelly, 1984). Another report indicated that tethering of sows suppressed
antibody synthesis to sheep red blood cell. It also resulted in a reduction in the amount of
antigen specific antibodies that were transmitted through the colostrum into the blood of the
piglets (Kelley, 1985).

One way to cope with these situations may be to improve the host defense mechanism
by boosting the immune system through the administration of immunomodulators.
Applications of irnmunostimulants seems promising as a means for the prevention or
treatment of various infections (Araki et al., 1993). In cattle it has been shown that Cr

behaves as an irnmunomodulator (Burton, 1995). Chang and Mowat (1992) reported that

86

supplemental Cr to beef cattle increased serum immunoglobulin levels during the growing
period. In another study, antibody production against human erythrocytes was increased
when Cr was supplemented during a stress period to weaned calves (Moonsie-Shaeger and
Mowat, 1993), and levels of antibody increased following vaccination (Burton et al., 1994).
During early lactation in dairy cows in which their diet was supplemented with chelated Cr,
a higher level of antibodies and a better in vitro blastogenesis response was observed when
the cows were challenged with a T cell mitogen (Burton et al., 1993). In other species it
appears that Cr also has some effect on the immune system. When stressed rats were
supplemented with dietary Cr mortality was reduced (Mertz and Roginski, 1969), and
Shroeder et al. (1965) reported that Cr supplementation reduced mortality from an epidemic
of pneumonia in female rats.

A common way of evaluating the influence of various factors on the immune system
is to measure the response of isolated peripheral blood lymphocytes to in vitro mitogen
stimulation. In the present study, to determine if Cr had any immunomodulator effect in the
cell mediated immune response of weanling pigs, we used phytohemagglutinin and
concanavalin A, two potent T cell mitogen and pokeweed mitogen and lipopolysaccharide
that are considered B cell mitogen. In this means we could evaluate the components of the
intrinsic immune system.

We found that dietary Cr supplementation to pigs reared in a crowded nursery
environment was ineffective in enhancing lymphocyte proliferation in the presence of optimal
concentration of all mitogen used. In a study with baby pigs, Van Heusken et al. (1994)

reports an enhanced lymphocyte proliferation in response to mitogen when Cr was

87

supplemented in the diet of nonstressed baby pigs, but there was no response in stressed baby
pigs. He also found that in vivo cellular immunity was not affected by Cr supplementation.
The addition of supplemental Cu, although tended to have a higher response, also failed to
influence a significant lymphocyte proliferation response to mitogen stimulation. These
results are consistent with the study of Arthington et al. (1995), who found no effect of Cu
and Zn supplementation on lymphocyte proliferation when stimulated by a mitogen.

In the first trial of this study there was an increase in mitogen induced lymphocyte
proliferation with increasing age for all the mitogen used. Similar results were found by
Hoskinson et al. (1990). However, in the second trial the proliferation was higher in the first
week and then tended to decrease with increasing age.

Neutrophils play a key role as part of the immune response to microbial infections.
Their major function is to rapidly kill bacteria and fungi before they multiply and spread
throughout the body. Nutritional deficiencies can affect the ability of neutrophils to
phagocytize and kill invading organisms. Sows receiving a selenium or vitamin E deficient
diet have a decreased phagocytic and microbicidal ability compared to those receiving a
nondeficient diet (W uryastuti, et al., 1993). Guinea pigs with a deficiency of vitamin C have
a reduction in the mobility and phagocytic activity of neutrophils ( Ganguly et al., 1976)
which was restored upon vitamin C supplementation. In this study we found that Cr or Cu
supplementation to a diet already containing 1.54 and 26 ppm of Cr and Cu respectively had
no effect on the phagocytic activity and the microbicidal ability of neutrophils of baby pigs.
On average all treatments showed an increase in the phagocytic activity and microbicidal

ability with increasing age. The measured values of phagocytic activity reported in this Study

88

are higher than those reported by Araki et al. (1994) in pigs of the same age, however a
direct comparison of our results with those of Araki et al. are difficult because of different
procedures. Our values for phagocytic activity and microbicidal ability are lower than the
values reported by Wuryastuty et a1. (1993) for sows. These differences may be due to a
difference in age. It has been reported that polymorphonuclear neutrophils have a decreased
bactericidal abilities in neonates compared with adult animals. In a study with horses,
Coignoul et al. (1984) found a significant reduction in myeloperoxidase-hydrogen peroxide-
halide activity by blood PMN in foals compared with mares. The myeloperoxidase hydrogen
peroxide system is extremely potent in the killing of a wide range of target cells and in the
inactivation of biological molecules of microbes. Similarly Hauser et al. (1986) reported that
iodination activity was lower in 4 to 5 week old than in 12 to 14 month old calves. In
another study blood PMN from calves also have deficient superoxide anion generation,

another important route in the neutrophil microbicidal system (Zwahlen et al., 1987).

IMPLICATIONS

The results of this study indicate that dietary chromium supplementation to a diet containing
1.54 ppm of chromium as provided by high chromium yeast had no apparent effect on Cu or
Zn retention of stressed baby pigs. It is also possible that for stressed baby pigs receiving a
com-soybean diet 22 ppm of Cu is not sufficient to provide for requirements. This study also
showed that Cr supplementation with or without Cu did not decrease the post weaning lag,
affect performance or the immune response of baby pigs reared in a crowded nursery
environment. However a better numerical ADG, ADFI and FIG ratio was observed in pigs

receiving supplemental Cr and a lower incidence of diarrhea was noted.

89

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“(mm—LL“

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It

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fl fibril L...

APPENDIX

113

Table 20. Proximal analysis of the basal diet compared to NRC recommendations.

II W Phrasal—EeLl—="

 

 

 

 

 

Nutrient Name Diet NRC Diet NRC
Composition Recommendation Comflgition Recommendation
Dry Matter % 89.82 89.10
Crude Protein % 19.64 20.00 20.29 18.00
Crude Fat % 2.79 2.63
‘Crude Fiber % 2.04 2.54
Calcium % 1.11 0.81 0.75 0.70
Phosphorus T % 0.86 0.65 0.69 0.60
Phosphorus A %. 0.60 0.39 0.39 0.32
Ash % 4.22 2.60
Swine ME Kcal 3215 3245 3267 3252

 

 

 

 

 

 

 

114

Table 21. Amino acid compositiona of the basal diet compared to NRC.

* , ' ‘ z * I mm... ‘ :"f' mm... 4 ..

 

   

AminoAcid' ' f M Diet   i, C 1' NRC   Diet ' 1’ H NRC
1 V . . . , Comw'tionr f : Recommendation, ,Coinr jitiOn I iRecommendationi

   

 

Lysine 1.26 1.15 1.22 0.95
amen-an ‘   «7642,52? wm : ‘. f ,w «m
Threonine 0.85 0.68 0.81 0.56
lsoleucine, , ’ 038 > i 0.65 W i037 0531' j'
Methionine + 0.65 0.59 0.73 0.48
Cysteine . g I p _ I V g '
Leucine 1.79 0.85 1.87 0.70
Hkfidinev ’   0.4975 4 ‘ 0.31 : g0.54~ i  8' 0151
Valine 1.02 0.68 1.06 0.56
beams... ’ : 0.90:5‘ ”0.94 i.‘  3'1.01   ?lo.7:1'v}y  
Tyrosine
A... e ” 01.21 3 4050 133040

 

 

 

 

 

 

“Estimated

115

Table 22. Vitamin composition“ of the basal diet compared to NRC.

IL Pkg; 3 Egg 92% 31.3%! ]|

 

 

 

Vitamin Diet NRC Diet NRC
Comw‘ifion Recommendation Comm. . ilion Recommendation,
Vit A 1000 IU 5.03 2.20 4.95 1.75
Vit D 1000 IU 0.99 0.22 0.99 0.20
Vit E 1000 IU 0.03 0.02 0.03 0.01
Vit K mg 3.41 0.04 3.43 0.05
Riboflavin mg 10.5 3.51 6.54 3.00
Panto. Acid mg 32.97 10.02 27.60 9.00
Niacin mg 48.05 15.03 48.15 12.51
Choline mg 1424 501 1325 400
Vit B12 [1g 38.70 17.54 29.70 15.00
Biotin #3 164.60 43.56 142.10 52.63
Folic Acid pg 453 305 407 294
Thiamine mg 3.32 1.00 3.37 1.00
Vit B6, mg 5.14 1.50 5.52 1.50

 

 

 

 

 

 

 

aEstimated

116

Table 23. Mineral composition“ of the basal diet compared to NRC.

II PhaseJEeed PW__-Eef-d_=1|

 

 

 

 

 

 

Mineral Diet NRC Diet NRC
Comwition Recommendation Com 'tion Recommendation
Sodium % 0.32 0.11 0.11 0.11
Chloride % 0.45 0.09 0.21 0.08
Potassium % 0.81 0.28 0.86 0.26
Magnesium % 0.15 0.04 0.16 0.04
Manganese mg/Kg 72.46 4.01 74.01 3.00
Iron mg/Kg 189.28 100.20 153.13 80.00
Copper mg/Kg 28.34 6.01 23.25 5 .00
Iodine mglKg 0.83 0.13 0.78 0.14
Zinc mg/Kg 141.86 100.20 141.81 80.00
Selenium mg/Kg 1.16 0.31 1.07 0.25

 

 

 

 

 

 

 

8by analysis

 

L—

 

117

Table 24. [3H]Thymidine uptake by unstimulated cells (CPM)?

M
g T1“ 12b '13c 14“ T1 T2 '13 T4
Day 0 3.08 2.60 2.78 2.67 2.57 2.76 2.70 2.82
Day 7 2.71 2.34 2.23 2.39 3.21 3.47 334 3.31
Day 14 2.55 2.33 2.61 2.46 3.29 3.77 4.11 4.11
Day 21 2.65 2.63 2.61 2.42 4.02 3.70 3.84 3.63
Day 28 2.27 2.42 2.47 2.42 2.96 3.15 3.37 3.24

Day 35 2.63 2.48 2.75 2.56 3.21 3.50 3.24 3.23

 

 

 

 

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

c Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation

d Basal diet with 0 ppm Cr and 250 ppm Cu supplementation

e Ratio between thymidine uptake value by stimulated and non stimulated cells.

Values are the log 10 of count per minutes.

118

Table 25. [3H]Thymidine uptake by concanavalin A stimulated cells (CPM)e .

 

 

an. . T1,“: . . T2”? .T3° 3 T44 ?*  T1 V 1273;713,"fang:

 

””0 234,. 3-31‘ 2'70, .3-‘374 ,V3-45H ,3-64. 93-96.. 3-51”.
9“?“ 4:09. .2-55 2-33 33" _.2-44_m3'54__ 3-36 400.
bay 21 f 3.03 3.0. 1 2.22; ; 2.41 “3405 “3.53 72.601 344
”a?“ _ .2-97 236 ‘2-54 w 4-50‘ H178 338., ,5-16. + 406.“
9435 ~ 4.03 ‘ i i 4.210; i _ 4... ‘ 449’ ‘ 2.57 '4... (4.19." 42.90--

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation

d Basal diet with 0 ppm Cr and 250 ppm Cu supplementation

e Ratio between thymidine uptake value by stimulated and non stimulated cells.

Values are the log 10 of count per minutes.

119

Table 26. [3H]Thymidine uptake by phytohemagglutinin stimulated cells (CPM)“.

 

 

j Day T1? { 1‘2" T3c .T4d'V'T1 T2T3T4

Day0 3.42 4.31 4.93 4.40 4.38 4.57 4.70 4.85 _

: nan. 5.16 if , 454 : ;. 4... '4-68. : 461441.76 . 4.7.4.7.
Day 14 4.22 4.73 4.67 4.44 5.10 p 5.44 p 5.52 5.41 .
nay 21’ _ 4.20 _ : .483 4.70-4.617,;1533 ~ 542'. 526510.
Day 28 . 4.74 4.58 4.89 5.10 5.04 5.09 5.02 4.91

 

Day 35 ' 4.76 4.60 “ 4.64 i 4.67 ' "4.55 34.88 [3.81][(4.73

 

 

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation

d Basal diet with 0 ppm Cr and 250 ppm Cu supplementation

6 Ratio between thymidine uptake value by stimulated and non stimulated cells.

Values are the log 10 of count per minutes.

 

120

Table 27. [3H]Thymidine uptake by pokeweed stimulated cells (CPM)e.

 

 

 

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

° Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation

d Basal diet with 0 ppm Cr and 250 ppm Cu supplementation

8 Ratio between thymidine uptake value by stimulated and non stimulated cells.

Values are the log 10 of count per minutes.

121

Table 28. Total pigs weight.

 

#6.“;
$7 T1“ T2b T3“ T4d T1 T2 T3 T4

Day 0 7.14 7.87 7.15 7.03 6.29 6.31 6.32 6.28
Day 7 8.28 9.11 8.27 8.20 6.93 6.72 7.07 7.17
Day 14 9.27 10.34 9.47 9.23 8.42 8.21 8.72 8.81
Day 21 11.48 12.85 11.44 11.52 10.28 10.20 10.61 10.95
Day 28 14.76 16.30 14.32 14.85 13.17 12.96 13.27 13.44

Day 35 18.45 20.66 18.22 18.83 17.22 16.69 17.03 16.84

 

 

 

 

 

 

 

 

 

 

 

 

 

a Basal diet 0 ppm Cr, 0 ppm Cu supplementation

b Basal diet with 0.50 ppm Cr and 0 ppm Cu supplementation

c Basal diet with 0.50 ppm Cr and 250 ppm Cu supplementation
d Basal diet with 0 ppm Cr and 250 ppm Cu supplementation

 

   

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