Trace Minerals in Animal Nutrition 2012 Arkansas Nutrition Conference Springdale, Arkansas

September 7, 2012

What a tangled web we weave when we discuss trace mineral and mineral nutrition. Ca

Fe

Cu

Na Mn

Zn

Mg Co

K

Cd P

(Adapted from Richards et al., 2007)

“With the myriad of small to medium sized Chinese manufacturers of trace mineral and mineral ingredients that constitute the suppliers of a standard large (shipload or barge load) tonnage shipment of product to the United States, along with possible comingling of various ingredients/suppliers at the port; it is virtually impossible to insure the quality of every pound of material received through the use of laboratory assays of this material, nor have adequate traceability of these ingredients, no matter how sophisticated or well equipped that laboratory may be.” ME. May, 2012

Theory #1 One of the main cost factors in broiler production today is oxidative stress during growout. Modern diets are seldom formulated to counteract this stress. Consider the following stressors: 1. The loss of antioxidant agents supplied by alfalfa and other ingredients that are no longer used in the modern diet because of their lack of nutrient density. 2. The loss of the antioxidant agents found in soybean meal and other protein sources that have been eliminated or significantly reduced in their addition rate due to their partial replacement by amino acids. 3. The stresses that modern production demands place on the birds, such as; ammonia exposure and bird density, heat stress, the reuse of multiple grow-out litter, early disease challenge and a hitherto unbelievably short growth cycle. It becomes rather obvious that the current antioxidant levels of the modern broiler diets can only marginally keep up with free radical production. Therefore, a reduction of free radical production, the sequestration of these radicals and the supplementation of diets with ingredients, elements, compounds or agents that support these two functions at adequate levels should be a goal of every modern broiler and turkey nutritionist.

From the mineral/trace mineral side of the diet ledger, when Se, I and Mg addition levels are increased above standard supplemental levels, it allows for more adequate production of glutathione peroxidase, (the #1 antioxidant in an animal’s body). Add to this the critical necessity for adequate dietary levels of the transition elements that are key to electron transfer both inside and outside of the cell (Mn, Zn, Cu and Fe, and Iodine), the proper and adequate supplementation of quality trace mineral and mineral compounds to poultry and turkey diets becomes of critical importance.

Our research has clearly demonstrated that, when Iodine is added at elevated nutritional levels (13 to 20 ppm), it helps to control the proliferation of pathogenic organisms and coccidiosis in growing broiler chickens to a comparable extent as currently utilized prophylactic use antibiotic programs, largely due to the activity of NIS (sodium iodide symporter). This is becoming more and more relevant as the FDA/USDA move forward with the elimination of the prophylactic use of antibiotics in broiler production.

Pro-oxidant

Antioxidant

The Concept of Peroxidative Balance According to Porta and Hartroft

Polyunsaturated lipids

Lipid Peroxidation =

Pro-oxidants

X Saturated lipids

Antioxidants

A free radical is defined as any atom, group of atoms, or molecules with one unpaired electron occupying an outer orbital. The one electron reduction of O2, called the univalent pathway, predominates radical production and the complete reduction of oxygen involves the addition of four electrons and four protons to each oxygen molecule. The resulting intermediates and the superoxide anion radical, O2− (O˙), hydrogen peroxide, H2O2, and the hyroxyl radical, OH −. Since these intermediates are very reactive, the modulation of this reactivity has been a dominating evolutionary pressure since O2 first appeared in the atmosphere. The Univalent Pathway for the Reduction of Molecular Oxygen and the enzymatic defense Mechanisms Available to Bypass and Prevent the Accumulation of Reactive Intermediates

Cytochrome c Oxidase Glutathione Peroxidase/Catalase O2

− + O2 − e + 2 H H2O2

Superoxide Dismutases

e − + H+

OH+ H2O

e − + H+

H2O

Putting it together: Selenium: Glutathione peroxidase (GHS-Px) and thioredoxin reductase (TR) are the most abundant antioxidant Se-containing proteins in mammals. Selenocysteine an integral part of both of these compounds. Selenium is primarily involved in the production of 6 ( six) different Se dependent enzymes in the glutathione peroxidase family. They differ in molecular weight, substrate specificity, cell distribution and perform a variety of functions. The primary selenium containing compound, i.e. selenocysteine ,is found in glutathione peroxidases, thioredoxin reductases, tetraiodothyronine 5’ deiodinases, formate dehydrogenases, glycine reductases and some hydrogenases. It has been dubbed as The 21st amino acid There are 25 to 30 active selenocysteine compounds (SeCys) among which are the iodothyronine deiodinases responsible for releasing iodine from T4 to create the active T3 form. Iodine: Caltalyzes the formation of glutathione peroxidase. Is dependent on triiodothyronine deiodinase ( a selenocysteine compound) for the release of iodine and conversion of thyroxine (T4) to triiodothyronine (T3) the active form of the thyroid hormone. Magnesium: Catalyzes the formation of glutathione and other metabolic functions that will be elucidated later in this presentation.

Green = Antioxidant and free radical protection

Cellular Free Radical Damage and Antioxidant Defense Mechanisms Superoxide radical (O-2) Super Oxide Dismutase and Ceruloplasmin

Black line = Free radical damage Iron (Fe++) catalyzed

Hydrogen peroxide

Diminished by Zinc and by Ceruloplasmin (Cu)

Glutathione Peroxidase and Catalyse

Scavangered by vitamins and A, E, C and Carotenoids

Hydroxyl radical Water Macromolecular damage

Protein SH-group Oxidation Zinc protects Cell dysfunction and cell death

Lipids

DNA strand breakage, etc.

Liperoxidation Protection by Zinc, Selenium, Vitamins A, E, C and Carotenoids Membrane damage

Other Macromolecules Chromosomal damage Zinc protects

Cell dysfunction and cell death

Mutagenesis Cell dysfunction

ENZYMES OF IMPORTANCE 1. 2. 3. 4. 5. 6. 7. 8. 9.

Cu,Zn SOD: Copper,zinc super oxide dismutase (1 & 3) Mn SOD: Manganese super oxide dismutase (2) Zn MTH: Zn-metallothionein GST: Glutathione S-transferase (Se, Mg) GPX: Glutathione peroxidase (Se, Mg, I2) GSH: Glutathione (reduced) (Se, Mg) GSSG: Glutathione (oxidized) (Se, Mg) GR: Glutathione reductase (Se, Mg) HMP: Hexose-monophosphate shunt (glucose-6-phosphate dehydrogenase, 6-phosphogluconic acid dehydrogenase) 11. CAT: Catalase (Fe)

ESSENTIAL NUTRIENTS INVOLVED IN RADICAL PRODUCTION AND SUBSEQUENT METABOLISM 1. Vitamin E –Tocopherol 2. Vitamin A (Retinol)

The body’s natural fat soluble oxidant Exact antioxidant function is Questionable. Carotenoids are actually pro-oxidants. 3. Ascorbic Acid Vitamin E recycling 4. Sulfur Amino Acids Glutathione, taurine, mercapturic acid formation 5. Copper Copper-zinc superoxide dismutase (cytoplasm) 6. Zinc Copper-zinc superoxide dismutase (cytoplasm) (membrane stabilization), Metallothionein 7. Manganese Manganese SOD, (mitochondrial protection) 8. Iron Catalase 9. Selenium Glutathione peroxidase and Sulphydryl protection 10. Riboflavin Co-enzyme for glutathione reductase 11. Nicotinic Acid NADPH (required for glutathione reductase) 12. Magnesium Glutathione synthesis 13. Phosphorus Hexose monophosphate shunt 14. Thiamine Transketolase (hexose monophosphate shunt) 15. Carotenes – Lutein, Zeaxanthin, β-cryptoxanthin, β-carotene, ά-carotene, Lycopene, Canthaxathin, Astaxanthin and Xanthophylls 16. Uric Acid

MULTILAYERED DEFENSE SYSTEM IN THE CELL DEFENSE LINE

TYPE OF DEFENSE

MAIN LOCATION

1st

Catalase, Mn - SOD

MT Matrix

2nd

Vitamin E – Membrane Bound

MT Inner Membrane

3rd

Cu, Zn SOD

MT Inner Membrane space and Cytoplasm

4th

Glutathione Peroxidase

Cytoplasm

5th

Ascorbic Acid, Glutathione, Uric Acid, Ceruloplasmin, Etc.

Serum, Tissues and Cytoplasm

MT = Mitochondrion SOD = Superoxide Dismutase

The Big Three Iodine – Selenium - Magnesium

Iodine Supplementary level in broiler diets should be 13 – 15 ppm. Breeder diets should be 4 ppm. Justification for these supplemental numbers may be found in the following slides.

Iodine The primary function of Iodine is as a component of the thyroid hormones, thyroxine (T4) and triiodothyronine (T3). These are tyrosine-based hormones produced by the thyroid gland primarily responsible for regulation of metabolism. The major form of thyroid hormone in the blood is thyroxine (T4), which has a longer half-life than (T3). The ratio of T4 to T3 released in the blood is roughly 20:1. Thyroxine is converted to “active” T3, (three to four times more potent than T4 ) within cells by selenium based deiodinases. These are further processed by decarboxylation and deiodination to produce iodothyronamine (T1a) and thyronamine (T0a). The thyronines act on nearly every cell in the body. They act to increase the basal metabolic rate, affect protein synthesis, help regulate long bone growth (synergy with growth hormone), neuronal maturation and increase the body’s sensitivity to catecholamines ( such as adrenaline) by permissiveness. The thyroid hormones are essential to proper development and differentiation of all cells of the human body. These hormones also regulate protein, fat and carbohydrate metabolism, affecting how cells use energetic compounds. They also stimulate vitamin metabolism. Numerous physiological and pathological stimuli influence thyroid hormone synthesis. Thyroid hormone leads to heat generation in humans. However, the thyronamines function via some unknown mechanism to inhibit neuronal activity; this plays an important role in the hibernation cycles of mammals and the moulting behavior of birds.

Because of our interest in iodine and our conviction that it was, in all likelihood, under-supplemented in animal feeds, we have run 6 scientific studies in broiler chickens using current and elevated iodine addition levels. In the first study, iodine was added to the birds drinking water. The other 5 studies were run using normal and elevated nutritional levels of iodine in the feed.

The summary data is to be found on the following slides.

Effect of Soluble Elemental Iodine in Drinking Water of Broilers Experiencing Necrotic Enteritis on Broiler Performance and Pre-harvest Intestinal Pathogenic Organisms. Study Number: 2002-SEM-14-B

Solution Biosciences, Inc. Salisbury, MD Ration Number SEM 14-1 (Control 1) SEM 14-2 (Control 2) SEM 14-3 (Control 3) SEM 14-4 SEM 14-5 SEM 14-6 SEM 14-7 SEM 14-8 SEM 14-9

Drinking Water Iodine (ppm) None None None 3 ppm 6 ppm 3 ppm 6 ppm 3 ppm 6 ppm

Challenge No No Yes No No Yes Yes Yes Yes

Feedgrade Antibiotic None BMD 50 g/ton BMD 50 g/ton None None None None BMD 50 g/ton BMD 50 g/ton

2520 birds total. Bacterial challenge includes Clostridium perfringens at eight (8) days of age and Salmonella spp., E. coli, Listeria monocytogenes and Campylobacter at 47 days of age, with harvest at 49 days of age.

Effect of Water Purification with Free Elemental Iodine at 3 and 6 ppm in the Drinking Water of Broiler Chickens on Performance in Normal and Stressed Conditions (Coccidial and Bacterial Challenge) Trial Day 0 to 42

Stressed Normal

Criterion

Control

Average Body Weight (g) Average Body Weight (lb) Stat 1 Feed Conversion Corrected Stat 1 Standard Deviation Stat 1 Coefficient of Variation

Stat 1 Mortality (%) Stat 1

Unchallenged Test Group 50 g/ton BMD 3 ppm I2 6 ppm I2

Stat 1

50 g/t BMD 50 g/t BMD 50g/ton BMD

& 3 ppm I2

& 6 ppm I2

1828.904 1888.387 1869.66 1891.655 1852.187 1844.28 1884.272 1868.283

1916.333

3 ppm I2 6 ppm I2

4.03

4.16

4.12

4.17

4.08

4.07

4.15

4.12

4.22

c

ab

abc

ab

bc

bc

abc

abc

a

1.778

1.758

1.759

1.745

1.762

1.758

1.725

1.748

1.723

c

bc

bc

ab

bc

bc

a

ab

a

155.03

149.78

183.45

189.97

129.46

195.95

155.93

b

ab

cd

bc

cd

d

a

d

b

8.48

7.94

9.85

8.8

9.91

10.34

6.87

10.49

8.15

b

ab

cd

bc

cd

d

a

d

ab

5.000

2.143

1.429

1.429

2.857

1.071

1.786

1.429

1.071

b

a

a

a

ab

a

a

a

a

Avg. Body Weight Gain (g) 1778.322 1838.709 Avg. Body Weight Gain (lb)

E. acervulina-E. maxima and C.Perfringens Challenge Group

184.47 165.97

1820.17 1841.788 1802.233 1794.39 1834.057 1818.140

1866.758

3.92

4.05

4.01

4.06

3.97

3.96

4.04

4.01

4.12

c

ab

abc

ab

bc

bc

abc

abc

a 21

42 Day Average Body Weight 1880 c

ab

abc

ab

bc

abc

a

1860 1840 1820 1800 1780 1760 1740

Optimal - Unchallenged

Stressed - Challenged

Control 50g/tBMD 3 PPM I2 6 PPM I2 50g/tBMD 3 PPM I2 6 PPM I2 50 Plus 3 50 Plus 6

42 Day Feed Conversion, Corrected 1.79

c

bc

ab

bc

a

ab

a

1.78 1.77 1.76 1.75 1.74 1.73 1.72 1.71

1.7

Optimal - Unchallenged

Stressed - Challenged

1.69 Control 50g/tBMD 3 PPM I2 6 PPM I2 50g/tBMD 3 PPM I2 6 PPM I2 50 Plus 3 50 Plus 6

42 Day Mortality 6 5

b

a

ab

a

4

3 2 1 Optimal - Unchallenged

Stressed - Challenged

0 Control 50g/tBMD 3 PPM I2 6 PPM I2 50g/tBMD 3 PPM I2 6 PPM I2

50 Plus 3 50 Plus 6

Effect of Water Purification with Free Elemental Iodine at 3 and 6 PPM in the Drinking Water of Broiler Chickens in Normal and Stressed Conditions (Coccidial and Bacterial Challenge) Stressed Normal

Criterion

Control

E. acervulina-E. maxima and C.Perfringens Challenge Group

Unchallenged Test Group 50 g/ton BMD 3 ppm I2 6 ppm I2

50g/ton BMD

50 g/t BMD

50 g/t BMD

3 ppm I2 6 ppm I2 & 3 ppm I2

& 6 ppm I2

Day 5 - Coccidiosis challenge; Day 8 Clostridium perfringens Average Lesion Score Day21 Stat 1 E. coli Count (per mL) Stat 1

Salmonella spp. Count/(mL) Stat 1 Listeria mono. Count/(mL) Stat 1 Campylobacter Count/(mL) Stat 1

0.232

0.250

0.339

0.036

1.536

1.339

0.411

0.804

0.161

bc

bcd

cd

a

g

f

d

e

ab

1586.4

1635.4

1328.6

1163.6

1766.8

1309.6

938.6

1288.2

850.7

de

e

cd

bc

e

cd

abc

c

a

145.4

136.9

100.9

34.2

134.0

110.9

65.9

111.9

58.7

d

d

c

a

d

c

b

c

b

152.2

142.0

110.3

52.0

153.9

119.1

66.9

131.6

65.5

e

de

b

a

e

bc

a

cd

a

124.7

123.1

119.3

107.0

125.2

121.6

112.0

127.8

108.3

cd

cd

abcd

a

cd

bcd

abc

d

ab

Wednesday, August 22, 2012

Southeastern, Marshall, Eastern and SEM Minerals, LP

25

21 Day Average Lesion Score 1.8

bc

bcd

cd

a

g

f

d

e

ab

1.6

1.4 1.2

1 0.8

0.6 0.4

0.2

Optimal - Unchallenged

Stressed - Challenged

0 Control 50g/tBMD 3 PPM I2 6 PPM I2 50g/tBMD 3 PPM I2 6 PPM I2

50 Plus 3 50 Plus 6

E. Coli Count (mL) 2000

de

e

cd

bc

e

cd

abc

c

a

1800 1600 1400 1200 1000 800 600

400 200

Optimal - Unchallenged

Stressed - Challenged

0 Control 50g/tBMD 3 PPM I2 6 PPM I2 50g/tBMD 3 PPM I2 6 PPM I2 50 Plus 3 50 Plus 6

Bacterial Counts (mL) Red = L. monocytogenes Blue = Salmonella spp. Green = Campylobacter

180

160 140 120 100 80 60

40

Optimal – Unchallenged

Stressed - Challenged

20 0 Control

50g/tBMD

3 PPM I2

6 PPM I2

50g/tBMD

3 PPM I2

6 PPM I2

50 Plus 3

50 Plus 6

Effect of Iodine Source and Level on Performance of Growing Broilers to 42 Days of Age When Placed Under Stress Field Conditions and Exposed to Specific Common Bacterial and Coccidial Challenges Study Number: 2002-SEM-14-B Solution Biosciences, Inc. Salisbury, MD Ration Number SEM 22-1 SEM 22-2 SEM 22-3 SEM 22-4 SEM 22-5 SEM 22-6 SEM 22-7

SEM 22-8 SEM 22-9 SEM 22-10 SEM 22-11 SEM 22-12

TEST MATERIAL (additives)2 Positive Control Groups CONTROL: BMD1 CONTROL: stEDDI Iodine source2 CONTROL: Calcium Iodate source Nutritional Test Groups stEDDI Iodine source Calcium Iodate source stEDDI Iodine source Calcium Iodate source Nutritional Test Stress Group stEDDI Iodine source stEDDI Iodine source stEDDI Iodine source stEDDI Iodine source stEDDI Iodine source

Added Levels of Iodine or BMD 50 g/ton BMD 1.0 ppm 1.0 ppm 2.0 ppm 2.0 ppm 3.0 ppm 3.0 ppm

5.0 ppm 7.0 ppm 9.0 ppm 11.0 ppm 13.0 ppm

Each ration was administered to six replicates of then chicks each for 0-42 days of age (or trial days 0-42). 1Bacitracin-MD (50g/ton) was used in the Control Treatment, only. 2520 total birds. All birds received Cocci-Vac for protection from coccidiosis. 2Control consists of a normal broiler starter, grower and finisher diet.

Performance Data – Day 42, 1 – 13 ppm Iodine in Feed Positive Controls BMD- STEDDI- CaIO350g/t 1ppm 1ppm

Criterion

EDDI and Calcium Iodate - Nutritional EDDI - Stress Levels STEDDI- CaIO3- STEDDI- CaIO3- STEDDI- STEDDI- STEDDI- STEDDI2ppm 2ppm 3ppm 3ppm 5ppm 7ppm 9ppm 11ppm

STEDDI13ppm

Average Body Weight (g) 1903.69 1851.95 1852.79 1865.89 1864.99 1874.09 1871.87 1886.9 1897.3 1900.26 1906.28 1911.49 Average Body Weight (lb) 4.19 4.08 4.08 4.11 4.11 4.13 4.12 4.16 4.18 4.19 4.20 4.21 STAT 1 ab d d cd cd bcd bcd abcd abc abc ab a Feed Conversion Corrected 1.832 1.903 1.910 1.878 1.879 1.867 1.861 1.849 1.846 1.844 1.824 1.815 STAT 1 Standard Deviation STAT 1

abc

Mortality (%)

f

def

def

cde

bcd

abcd

abcd

abcd

183.71 183.44 191.83 170.72 180.13 186.86 183.78 177.79 193.87 182.12 a

ab

a

186.02 180.16

a

a

a

a

a

a

a

a

a

a

a

9.91

10.34

9.15

9.66

9.97

9.82

9.43

10.22

9.60

9.77

9.43

a

a

a

a

a

a

a

a

a

a

a

a

1.88

1.72

2.03

1.88

1.88

1.88

2.19

1.56

1.72

2.03

1.88

1.88

Coefficient of Variation 9.65 STAT 1

ef

STAT 1 a a a a a a a a a a a a Avg. Body Weigh Gain (g) 1858.76 1807.01 1807.81 1820.95 1820.02 1829.15 1826.94 1841.99 1852.4 1855.31 1861.36 1866.54 Avg. Body Weight Gain (lb) 4.09 3.98 3.98 4.01 4.01 4.04 4.02 4.06 4.08 4.09 4.10 4.11 STAT 1

ab

d

d

cd

cd

bcd

bcd

abcd

abc

abc

ab

30

a

Challenge and General Data – Day 42 Positive Controls BMD- STEDDI- CaIO350g/t 1ppm 1ppm

Criterion

Lesion Score

0.13

EDDI and Calcium Iodate - Nutritional EDDI - Stress Levels STEDDI- CaIO3- STEDDI- CaIO3- STEDDI- STEDDI- STEDDI- STEDDI- STEDDI2ppm 2ppm 3ppm 3ppm 5ppm 7ppm 9ppm 11ppm 13ppm

0.49

0.56

0.39

0.33

0.38

0.33

0.3

0.28

0.16

0.14

0.08

STAT 1 a Litter Condition Ideal 6/10

ef

f

de

d

de

d

cd

bcd

abc

ab

a

2/10

3/10

4/10

5/10

5/10

6/10

7/10

8/10

8/10

9/10

8/10

Too Dry

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

1/10

2/10

Too Wet Feathering Score

4/10

8/10

7/10

6/10

5/10

5/10

4/10

3/10

2/10

2/10

0/10

0/10

3.10

3.12

3.10

3.06

3.12

3.13

3.20

3.13

3.11

3.10

3.15

3.11

STAT 1 ab Flightiness Score 1.00

ab 0.60

ab 1.40

b 1.00

ab 1.00

ab 1.20

a 1.00

ab 1.10

ab 1.10

ab 1.50

ab 1.10

ab 0.80

STAT 1 ab Skin Pigmentation Redness Value 1.63

b

ab

ab

ab

ab

ab

ab

ab

a

ab

ab

1.61

1.63

1.63

1.63

1.64

1.63

1.63

1.63

1.63

1.63

1.62

STAT 1 a a a a a a a a a a a a Yellowness Value 14.23 11.156 11.154 15.18 15.202 15.148 15.297 15.235 15.284 16.201 17.014 17.181 STAT 1 c d Lightness Value 65.65 55.49 STAT 1

a

b

d 55.42

b 65.37

b 65.52

b 65.66

b 65.45

b 65.8

b

a

a

a

a

a

b b 65.63 65.29 a

a

a 65.65

a 65.71

a

a 31

42 Day Average Body Weight 1920

ab

d

cd

bcd

abcd

abc

ab

1910 1900

1890 1880 1870 1860 1850 1840 1830 1820

Positive Controls

Nutritional Levels

Nutritional-Stress Levels

a

42 Day Feed Conversion - Corrected 1.92

abc

ef

f

def

cde

bcd

abcd

ab

1.9 1.88 1.86 1.84 1.82 1.8 1.78 1.76

Positive Controls

Nutritional Levels

Nutritional-Stress Levels

a

Lesion Score – Day 42 0.6 0.5 0.4 0.3 0.2 0.1 0

a

ef

f

de

d

de

d

cd

bcd

abc

ab

a

Ideal Litter Condition – Day 42 1 9/10 8/10 7/10 6/10 5/10 4/10 3/10 2/10 1/10 0

Feathering Score – Day 42 ab 3.25 3.20 3.15 3.10

3.05 3.00 2.95

b

ab

a

ab

Flightiness Score – Day 42 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00

ab

b

ab

a

ab

Skin Pigmentation - Yellowness Value – Day 42 20 18 16 14 12 10

8 6 4 2 0

c

d

b

a

Skin Pigmentation – Lightness Value – Day 42 80 70 60 50 40 30 20 10 0

a

b

a

Bacterial Challenge Data – Day 42 EDDI and Calcium Iodate Positive Controls Nutritional EDDI - Stress Levels BMD- STEDDI CaIO3- STEDDI- CaIO3- STEDDI CaIO3- STEDDI STEDDI STEDDI STEDDI- STEDDI50g/t -1ppm 1ppm 2ppm 2ppm -3ppm 3ppm -5ppm -7ppm -9ppm 11ppm 13ppm

Criterion

Crop Total Aerobic Cnt. (log cfu's) STAT 1

4.54

6.28

6.95

6.36

6.38

6.11

6.08

5.68

5.61

4.79

4.76

4.16

ab

de

e

de

de

de

cde

bcde

bcd

abc

ab

a

5.95

5.73

5.50

5.68

5.50

5.53

5.10

5.19

5.16

4.79

4.32

a

c

c

bc

bc

bc

bc

abc

abc

abc

ab

a

3.97

5.44

5.18

5.35

5.60

5.24

4.32

4.26

4.48

4.20

4.08

3.74

ab

c

c

c

c

c

ab

ab

b

ab

ab

a

4.78

4.69

4.92

4.77

4.78

4.77

4.71

4.67

4.70

4.76

4.70

a

a

a

a

a

a

a

a

a

a

a

Crop Salmonella Content 4.30 (log cfu's)

STAT1

Crop E. coli Content (log cfu's) STAT 1

Crop Campylobacter Cnt 4.54

(log cfu's)

STAT1

a

Crop Total Aerobic Content – Post Challenge 8 7 6 5

log 4 cfu’s 3 2 1

0

Blue – Total Aerobic Count Red – Salmonella Count Green – E. coli Count Purple – Campylobacter Count

Crop Total Aerobic Content – Post Challenge 8 7 6 5 log Cfu’s4 3 2 1 0

ab

de

e

de

cde

bcde

bcd

abc

ab

a

Crop Salmonella Content 7.00 6.00 5.00 4.00 log Cfu’s 3.00 2.00 1.00 0.00

a

c

bc

abc

ab

a

Crop E. coli Content 6.00 5.00 4.00 log Cfu’s 3.00 2.00 1.00 0.00

ab

c

ab

b

ab

a

Effect of Iodine Source on Goitrogenic Response in 12 and 21 Day-Old Broiler Chicks Reared in Cages Study Number: 2009-SEM-24-B Completed December 14, 2009 Solution Biosciences, Inc. Salisbury, MD Ration Number

TEST MATERIAL (additives)

Negative CONTROL2 SEM 24-1 SEM 24-2 SEM 24-3 SEM 24-4 SEM 24-5 SEM 24-6 SEM 24-7 1Each

Added Levels (Dietary Iodine, ppm) No added Iodine (Goitrogenic Basal Diet)

Test Groups1

EDDI (79.6% Iodine) stEDDI Stabilized EDDI (79.6% iodine) EDDI (79.6% Iodine) stEDDI Stabilized EDDI (79.6% iodine) EDDI (79.6% Iodine) stEDDI Stabilized EDDI (79.6% iodine)

1.0 ppm 1.0 ppm 10.0 ppm 10.0 ppm 20.0 ppm 20.0 ppm

ration will be administered to eight replicates of ten chicks each for 0-21 days of age. 560 total chicks. 2Control consists of a normal broiler starter goitrogenic-type diet (without added iodine)

IMPORTANT! Effect of Iodine Source on Goiterogenic Response in 12 and 21 Day-Old Broiler Chicks Reared in Cages Trt. #1

Trt. #2

Trt. #3

Trt. #4

Trt. #5

Trt. #6

Trt. #7

EDDI

STEDDI-67

EDDI

STEDDI-67

EDDI

STEDDI-67

Neg. Cont.

1 ppm

1 ppm

10 ppm

10 ppm

20 ppm

20 ppm

618.67

629.40

631.53

648.57

654.55

667.3

676.55

d

d

d

cd

bc

ab

a

1.536

1.496

1.482

1.447

1.424

1.424

1.406

e

d

cd

bc

ab

ab

a

573.40

583.60

586.23

603.37

609.97

622.33

630.63

d

d

d

cd

bc

ab

a

18.22

14. 08

12.90

9.35

7.90

7.51

6.18

d

c

c

b

ab

ab

a

40.47

31.82

30.68

20.17

17.41

10.15

9.19

e

d

d

c

b

a

a

0.00

21.4%

24.2%

50.2%

57.0%

Performance Data Average Body Weight (g) Day 21 Stat1 Feed Conv. Corrected Day 0-21

Stat1 Avg. Body Wt. Gain Day 0-21 Stat1

Thyroid Data Thyroid Wt. (mg/100BW) Day 12 Stat1

Thyroid Wt.(mg/100BW)Day21 Stat1

Percentage Reduction

74.9% 77.5%

Average Body Weight Gain – Day 0 - 21 640

d

cd

bc

ab

ab

630 620 610 Grams

600 590 580 570 560 550 540 Neg. Cont. E- 1 ppm S- 1 ppm E- 10 ppm S-10 ppm E- 20 ppm S-20 ppm

Feed Conversion, Corrected Day 0 - 21 1.55

e

d

cd

bc

ab

a

Feed/Gain Ratio

1.5

1.45

1.4

1.35

1.3

Neg. Cont. E- 1 ppm S- 1 ppm E- 10 ppm S-10 ppm E- 20 ppm S-20 ppm

Average Thyroid Weight (mg/100g BW) Day 21 45

e

d

d

c

b

a

Thyroid Weight (mg/100g Body Weight

40 35 30 25 20 15 10 5

0

%Reduction 0.00

21.4%

Neg. Cont. E- 1 ppm

24.2%

50.2%

57.0%

74.9%

77.5%

S- 1 ppm E- 10 ppm S-10 ppm E- 20 ppm S-20 ppm

In order for the remaining studies to make a modicum of sense, I will jump to a Magnesium Study that was completed on June 13, 2010.

Effect of Magnesium Level on Broiler Chick Performance and Bone Strength When Raised Under Commercial Grow-out Conditions Trial: 2010-SEM-26-B Summary Date: June 13, 2010 Trial Conducted by: AHPharma, Inc. of Salisbury, Maryland

Ration Number SEM 26-1 SEM 26-2 SEM 26-3 SEM 26-4 SEM 26-5 SEM 26-6 SEM 26-7 1Each

Test Material (additives)1 None (Control) 1 lb/ton Feedox® Poultry Magnesium Blend 2 lb/ton Feedox® Poultry Magnesium Blend 3 lb/ton Feedox® Poultry Magnesium Blend 4 lb/ton Feedox® Poultry Magnesium Blend 5 lb/ton Feedox® Poultry Magnesium Blend 6 lb/ton Feedox® Poultry Magnesium Blend

Added Dietary Mg Levels Str Gro Fin None None None 0.028 0.028 0.028 0.056 0.056 0.056 0.084 0.084 0.084 0.112 0.112 0.112 0.140 0.140 0.140 0.168 0.168 0.168

treatment was fed to 10 replicates of 70 mixed sex broilers. 4900 total birds .

Corrected -Total Added Magnesium and Dietary Ca:Mg and P:Mg Ratios Added Magnesium Element Total Starter Mg

0.000 0.184

0.028 0.212

0.056 0.240

0.084* 0.268

0.112 0.296

0.140 0.324

0.168 0.352

Diet Ca Ca:Mg

0.90 4.89

4.25

3.75

3.36

3.04

2.78

2.56

Diet P

0.45

P:Mg

2.17

1.89

1.67

1.49

1.35

1.23

1.14

Total Grower Mg

0.193

0.221

0.249

0.277

0.305

0.333

0.361

Diet Ca

0.85

Ca:Mg Diet P

4.04 0.42

3.85

3.41

3.07

2.79

2.55

2.36

P:Mg

2.18

1.90

1.68

1.52

1.38

1.26

1.16

Total Finisher Mg Diet Ca Ca:Mg

0.202 0.75 3.71

0.230

0.258

0.286

0.314

0.342

0.370

3.36

2.91

2.62

2.39

2.19

2.03

Diet P

0.38

P:Mg

1.88

1.65

1.47

1.33

1.21

1.11

0.19

*Appears to be optimum level. NRC – Mg = 0.06; Ca = 1.0/0.9/0.8; P = 0.45, 0.35, 0.30.

Performance Day 0 - 42 Treatment Treatment Treatment Treatment Treatment Treatment Treatment 1 2 3 4 5 6 7 Control No MgO

Average Body Wt. (g) Day 42 STAT Feed Conversion Corrected, Day 0-42 STAT Standard Deviation, Day 42 STAT Coefficient of Variation, Day 42 STAT Mortality %, Day 0 - 42 STAT Average Body Wt. Gain (g), Day 042 STAT

1 lb. MgO 2 lbs. MgO 3 lbs. MgO 4 lbs. MgO 5 lbs. MgO 6 lbs. MgO 0.028% Mg 0.056% Mg 0.084% Mg 0.112% Mg 0.140% Mg 0.168% Mg

2184.46

2203.62

2224.87

2247.48

2250.97

2250.47

2250.15

b

ab

ab

a

a

a

a

1.869

1.859

1.838

1.838

1.837

1.836

1.834

b

ab

a

a

a

a

a

126.48

126.06

130.69

117.18

121.13

125.82

124.55

a

a

a

a

a

a

a

5.79

5.72

5.87

5.22

5.38

5.59

5.54

a

a

a

a

a

a

a

1.83

1.33

0.67

0.33

0.67

0.5

0.67

a

a

a

a

a

a

a

2139.54

2158.71

2179.93

2202.49

2206.00

2205.52

2205.19

a

a

a

a

a

a

a

Flightiness Score Flightiness Score, Day 21 STAT Flightiness Score, Day 42 STAT

Treatment Treatment Treatment Treatment Treatment Treatment Treatment 1 2 3 4 5 6 7 Control 1 lb. MgO 2 lbs. MgO 3 lbs. MgO 4 lbs. MgO 5 lbs. MgO 6 lbs. MgO No MgO 0.028% Mg 0.056% Mg 0.084% Mg 0.112% Mg 0.140% Mg 0.168% Mg 0.50 0.40 0.60 0.50 0.70 0.70 0.80 a a a a a a a 0.90 1.00 0.80 0.90 1.10 1.00 0.90 a a a a a a a

Treatment Treatment Treatment Treatment Treatment Large Intestine Fecal Moisture Treatment 1 Treatment 2 3 4 5 6 7 Control 1 lb. MgO 2 lbs. MgO 3 lbs. MgO 4 lbs. MgO 5 lbs. MgO 6 lbs. MgO No MgO 0.028% Mg 0.056% Mg 0.084% Mg 0.112% Mg 0.140% Mg 0.168% Mg Large Intestine Fecal Moisture (Day 21) 66.68 66.08 66.18 66.82 67.28 68.50 69.70 STAT a a a a ab ab b Large Intestine Fecal Moisture (Day 42) 75.62 75.88 75.78 75.25 76.71 77.71 80.58 STAT a a a a a ab b

Treatment Treatment Treatment Treatment Treatment Treatment Treatment 1 2 3 4 5 6 7 Control 1 lb. MgO 2 lbs. MgO 3 lbs. MgO 4 lbs. MgO 5 lbs. MgO 6 lbs. MgO No MgO 0.028% Mg 0.056% Mg 0.084% Mg 0.112% Mg 0.140% Mg 0.168% Mg Tibia Bone Ash (21 days) 46.55 46.95 47.28 47.75 47.76 47.71 47.92 STAT c bc b a a a a Tibia Bone Ash (42 days) 48.43 49.29 50.61 51.93 51.86 51.86 51.85 STAT c bc ab a a a a Tibia Breaking Strength (21 days) 9.72 10.03 10.21 10.25 10.39 10.32 10.38 STAT d c b ab a ab a Tibia Breaking Strength (42 days) 17.2 17.46 17.51 17.55 17.8 17.85 17.83 STAT d cd bcd abc ab a a

42-Day Average Body Weight Gain (g) 2220 2200

Grams

2180 2160 2140 2120 Mg-% Add 2100 STAT

C

0.028

a

a

1

2

0.056

a

3

0.084

a

4

0.112

a

5

0.140

a

6

0.168

a

7

Note: Fecal Moisture was only significantly affected by the 0.168% Mg addition. Flightiness was not significantly affected at any Mg addition level.

53

42-Day Tibia Bone Ash (g)

52 51 Grams

50 49 48 47 46 Mg-% Add 1C STAT c

0.028 2

bc

0.056 3

b

0.084 4

a

0.112 5

a

0.140 6

a

0.168 7

a

42-Day Tibia Breaking Strength 18 17.8 17.6 17.4 17.2 17 Mg-% Add 16.8 STAT

C

0.028

0.056

0.084

0.112

0.140

0.168

1d

cd2

bcd 3

4abc

5ab

6a

a7

Final Live Performance Data Summary Trial: SEM 11 Summary Date: March 30, 2011 Trial Conducted by: Southern Poultry Research, Inc. Treatment Design Treatment Number1 1- Control 2 3 4 5 6

7

1Each

Starter xxx BMD (50 g/ton) stEDDI - 15 ppm I BMD (50 g/ton) Mg -0.08% from MgO stEDDI - 15 ppm I Mg -0.08% from MgO BMD (50 g/ton) stEDDI - 15 ppm I Mg -0.08% from MgO BMD (50 g/ton) stEDDI - 15 ppm I

Grower xxx BMD (50 g/ton) stEDDI - 15 ppm I BMD (50 g/ton) Mg -0.08% from MgO stEDDI - 15 ppm I Mg -0.08% from MgO BMD (50 g/ton) stEDDI - 15 ppm I Mg -0.08% from MgO BMD (50 g/ton) stEDDI - 15 ppm I

treatment was fed 8 replicates of 50 male broiler chickens. 2800 total birds.

Finisher xxx Stafac (20 g/ton) stEDDI - 15 ppm I Stafac (20 g/ton) Mg -0.08% from MgO stEDDI - 15 ppm I Mg -0.08% from MgO Stafac (20 g/ton) stEDDI - 15 ppm I Mg -0.08% from MgO Stafac (20 g/ton) stEDDI - 15 ppm I

An Evaluation of Feeding stEDDI Iodine Source to Male Broilers - 42 Day Data

Day 42 Feed Consumption STAT Adusted Feed Conversion STAT Average Live Weight (kg.) STAT % Mortality STAT % Foot Ash (42-day) STAT

Treament 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5 Treatment 6 BMD 50g/t BMD 50g/t BMD 50g/t (S/G) x (S/G) x (S/G) Stafac 20g/t Stafac 20g/t Stafac 20g/t (F) x (F) x (F) stEDDI 15 stEDDI 15 stEDDI 15 Control x ppm I x ppm I ppm I MgO - .08% MgO - .08% MgO - .08% x x Mg Mg Mg

Treatment 7 BMD 50g/t (S/G) Stafac 20g/t (F) stEDDI 15 ppm I x

215.46

215.61

216.17

213.88

211.12

208.26

213.84

ab

a

a

ab

bc

c

ab

1.859

1.813

1.805

1.820

1.827

1.766

1.820

a

b

b

b

ab

c

b

2.313

2.395

2.407

2.352

2.310

2.394

2.345

c

ab

a

abc

c

ab

bc

2.75

3.75

3.25

2.75

2.50

4.50

2.25

a

a

a

a

a

a

a

13.935

13.736

13.933

13.427

13.318

13.906

13.479

a

ab

a

ab

bc

a

b

42-Day Average Live Weight (kg.) 2.42 2.4

kilograms

2.38 2.36

2.34 2.32 2.3 2.28

Treatment

2.26

Control

1 STAT

c

BMD Stafac 2X X

X X stEDDI-15 3 X

ab

a

BMD X BMD BMD Stafac X Stafac Stafac stEDDI-15 stEDDI-15 stEDDI-15 4X 5 6 7 Mg-.08% Mg-.08% Mg-.08% X

abc

c

ab

bc

42-Day Adjusted Feed Conversion 1.88 1.86 1.84

Ratio

1.82 1.8 1.78 1.76 1.74 1.72 Treatment

Control

1

BMD Stafac X X2

X X stEDDI-15 X3

BMD Stafac X Mg-.08% 4

a

b

b

b

1.7 STAT

X BMD BMD X Stafac Stafac stEDDI-15 stEDDI-15 stEDDI-15 Mg-.08% Mg-.08% X7 5 6

ab

c

b

An Evaluation of Feeding stEDDI Iodine Source to Male Broilers - 42 Day Data. stEDDI @ 15 ppm is equal to BMD @ 50g/ton and Stafac @ 20g/ton in improving feed conversion No Statistically Significant Synergistic Effect. Treament 1

Day 42 Feed Consumption STAT Adusted Feed Conversion STAT Average Live Weight (kg.) STAT % Mortality STAT % Foot Ash (42-day) STAT

Treatment 3 x

Treatment 2 BMD 50g/t (S/G)

Treatment 7 BMD 50g/t (S/G)

Control

x stEDDI 15 ppm I

Stafac 20g/t (F) x

Stafac 20g/t (F) stEDDI 15 ppm I

215.46 ab 1.859 a 2.313 c 2.75 a 13.935 a

x 216.17 a 1.805 b 2.407 a 3.25 a 13.933 a

x 215.61 a 1.813 b 2.395 ab 3.75 a 13.736 ab

x 213.84 ab 1.820 b 2.345 bc 2.25 a 13.479 b

42-Day Average Live Weight (kg.) - Iodine Effect 2.42 2.4

Kilogram

2.38 2.36 2.34 2.32 2.3 2.28 Treatment

2.26

STAT

Control X 1X

c

stEDDI-15ppm I X X2

a

X BMD 50g/t(S/G) Stafac 3 20g/t(F)

ab

stEDDI-15ppm I BMD 50g/t(S/G) Stafac420g/t(F)

bc

42-Day Adjusted Feed Conversion - Iodine Effect

Ratio

1.87 1.86 1.85 1.84 1.83 1.82 1.81 1.8 1.79 1.78 Treatment 1.77 STAT

Control X 1X a

stEDDI-15ppm I X 2X b

X BMD 50g/t(S/G) Stafac320g/t(F) b

stEDDI-15ppm I BMD 50g/t(S/G) 4 Stafac 20g/t(F) b

Final Live Performance Data Summary Trial: 2012-SEM-29-B Summary Date: March 5, 2012 Trial Conducted by: AHPharma, Inc. of Salisbury, Maryland Treatment Design Treatment Number1,2,3 1- Control 2 3 4 5 6 7 8 9 10 1Each

Basal Ration Type Corn-Soy Basal Diet Corn-Soy Basal Diet Corn-Soy Basal Diet Corn-Soy-20% DDGS Diet Corn-Soy Basal Diet Corn-Soy Basal Diet Corn-Soy-20% DDGS Diet Corn-Soy Basal Diet Corn-Soy Basal Diet Corn-Soy-20% DDGS Diet

Added Selenium

Added Iodine Level

Level (%) 0.30 0.30 0.30 0.30 0.60 0.60 0.60 0.90 0.90 0.90

(ppm) 1.2 4.0 13.0 13.0 4.0 13.0 13.0 4.0 13.0 13.0

treatment will be fed 8 replicates of 44 mixed-sex broilers. 3520 total birds. 3A basal (with enough feed for the entire treatment) will be mixed first and then premix mixture will be added.

Trial: 2012-SEM-29-B Control

Through Day 49 Average Body Wt. (g) STAT Feed Conversion Corrected

STAT Standard Deviation

STAT Coefficient of Variation

STAT Mortality % STAT Average Body Wt. Gain (g) STAT

Treatment 2 Treatment 3 Treatment 4 Treatment 5 Treatment 6 Treatment 7 Corn/Soy/DD Corn/Soy/DD Corn/Soy Corn/Soy Corn/Soy GS Corn/Soy Corn/Soy GS 1.2 I - 0.3 13.0 I - 0.6 Se 4.0 I - 0.3 Se 13.0 I - 0.3 Se 13.0 I - 0.3 Se 4.0 I - 0.6 Se Se 13.0 I - 0.6 Se 2530.1

2529.8

2569.8

2574.3

2570.2

2606.3

2612.4

c

c

bc

abc

bc

abc

abc

2.000

1.989

1.970

1.967

1.988

1.960

1.951

b

ab

ab

ab

ab

ab

ab

304.41

325.81

345.06

322.64

334.42

323.30

330.48

ab

bc

c

abc

c

abc

bc

12.06

12.87

13.43

12.52

13.00

12.39

12.65

a

a

a

a

a

a

a

3.00

3.50

3.00

3.00

2.75

3.00

2.50

a

a

a

a

a

a

a

2483.20

2482.80

2523.00

2527.50

2523.40

2559.50

2565.40

c

c

bc

abc

bc

abc

abc

Trial: 2012-SEM-29-B, continued

Through Day 49 Average Body Wt. (g) STAT Feed Conversion Corrected STAT Standard Deviation STAT Coefficient of Variation STAT Mortality % STAT Average Body Wt. Gain (g) STAT

Control

Treatment 8

Treatment 9

Treatment 10

Corn/Soy

Corn/Soy

Corn/Soy

Corn/Soy/DDGS

1.2 I - 0.3 Se

4.0 I - 0.9 Se

13.0 I - 0.9 Se

13.0 I - 0.9 Se

2530.1

2615

2654.6

2660

c

abc

ab

a

2.000

1.967

1.944

1.935

b

ab

ab

a

304.41

303.64

318.65

296.52

ab

ab

abc

a

12.06

11.63

12.01

11.14

a

a

a

a

3.00

2.50

2.75

2.75

a

a

a

a

2483.20

2568.10

2607.80

2613.10

c

abc

ab

a

49-Day Average Body Weight Gain (g) 2650

Grams

2600 2550 2500

2450 2400 Treatment STAT

Control C/S C/S C/S/DD C/S C/S C/S/DD C/S C/S C/S/DD 1.2/.3Se 4.0/.6 1 4.0/.3 2 13.0/.3 3 13.0/.3 4 5 13.0/.6 6 13.0/.6 7 4.0/.9 8 13.0/.9 9 13.0/.9 10

c

c

bc

abc

bc

abc

abc

abc

ab

a

49-Day Feed Conversion Corrected 2.020 2.000

Ratio

1.980 1.960 1.940 1.920 1.900 C/S Treatment C/S C/S C/S/DD C/S C/S C/S/DD C/S C/S C/S/DD I/Se 1.2/.3Se 1 4.0/.3 2 13.0/.3 3 13.0/.3 4 4.0/.6 5 13.0/.6 6 13.0/.6 7 4.0/.9 8 13.0/.9 9 13.0/.9 10 STAT b ab ab ab ab ab ab ab ab a

Important Note: These data indicate that adding Iodine at 1.0 to 4.0 ppm does not support maximum growth and maximum feed efficiency. When iodine is increased to 13 ppm, maximum performance at each selenium addition level was achieved. There appears to be a definite synergistic effect that is realized when iodine and selenium addition levels are concurrently increased, with optimum performance being achieved by adding 13 ppm iodine and .9 ppm selenium from sodium selenite. These data support the supposition that Iodine and Selenium are interdependent in overcoming the oxidative stress the birds encounter during grow-out.

The following Study was scheduled for statistical analysis during the week of August 12, 2012; therefore the final report was not available on the date this presentation was compiled. Slides will be added in order to present the data from this study at the September Conference.

Treatment Number 1-Negative Control Not Challenged 2

Basal Ration Type Corn-Soy Basal Diet Negative Control NOT Challenged Corn-Soy Basal Diet NOT Challenged

Antibiotic & Level (g/ton of complete feed) None

(Iodine)

3

Corn-Soy Basal Diet NOT Challenged

(Iodine)

4

Corn-Soy Basal Diet NOT Challenged

Bacitracin-MD (50 g/ton).

5

Corn-Soy Basal Diet NOT Challenged

6

Corn-Soy Basal Diet NOT Challenged

Tylan or Tylosin Phosphate (20 g/ton). Virginiamycin (20 g/ton).

7-Negative Control Challenged 8

Corn-Soy Basal Diet Negative Control Challenged

None

Corn-Soy Basal Diet Challenged

(Iodine)

9

Corn-Soy Basal Diet Challenged

(Iodine)

10

Corn-Soy Basal Diet Challenged

Bacitracin-MD (50 g/ton).

Corn-Soy Basal Diet Challenged

Tylan or Tylosin Phosphate (20 g/ton). Virginiamycin (20 g/ton).

11

12

Corn-Soy Basal Diet Challenged

Litter Type Administration (See Footnote 1) New/Fresh/ Uncontaminated NOT Challenge New/Fresh/ Uncontaminated NOT Challenge New/Fresh/ Uncontaminated NOT Challenge New/Fresh/ Uncontaminated NOT Challenge New/Fresh/ Uncontaminated NOT Challenge New/Fresh/ Uncontaminated NOT Challenge Built-up Litter Bedding & Challenged 1 Built-up Litter Bedding & Challenged 1 Built-up Litter Bedding & Challenged 1 Built-up Litter Bedding & Challenged 1 Built-up Litter Bedding & Challenged 1 Built-up Litter Bedding & Challenged 1

Added Iodine Level (ppm) None

15

20

None

None

None

None

15

20

None

None

None

What is the Mechanism of Delivery of Iodine to the Gastric Lumen of the Intestine? Answer, or at least a part of it! Sodium Iodide Symporter (NIS) in Gastric Mucosa: Gastric Iodide Secretion Malin Josefsson, University of Lund; Malmö, Sweden, 2009.

Iodide is actively transported from the bloodstream into the gastric juice and some iodide accumulation occurs in the gastric wall, but no uptake of iodide takes place in the gastric lumen. In 1996, it was revealed that, in the thyroid gland, iodide is actively transported into the thyrocyte by NIS. Later, NIS was also found to be present in large amounts in the gastric mucosa, where it is located basolaterally in the surface epitehlail cells. Iodine is actively transported from the bloodstream into the gastric lumen, but not in the opposite direction. Iodide transport over the gastric mucosa is attenuated by the selective competitive NIS inhibitor perchlorate, and also by ouabain, and inhibitor of Na +/K+-, ATPase which powers NIS transport. This gastric iodide secretion is, to a large extent, mediated by NIS. The functional role of NIS in the gastric mucosa is uncertain, but several theories have been put forward. These include mediating the recirculation of iodine, as well as securing the presence of iodide in the stomach for antimicrobial or antioxidative purposes. Gastric iodide secretion may also be a protecting mechanism against developing gastric cancer. Gastric NIS has further been suggested to be an important protein for transporting anions other than iodide, i.e. nitrate. This is of interest in that NO3¯ is reduced to nitrite (NO2¯) by bacterial enzymes and, in an acidic environment, the non-enzymatically reduced to nitric oxide (NO), a powerful antimicrobial agent. Thus both iodide and NO3¯ may play important roles in the defense against microbes.

In addition, it was also indicated that the presence of iodide enhances the antimicrobial effect of NO. Interestingly, an entero-salivary recirculation of NO3¯ has been suggested by several groups and the salivary glands are, together with the thyroid, gastric mucosa and lactating mammary gland, the locations in which NIS is expressed in considerable amounts. Using the salivary glands as an example of NIS activity, it can be noted that the eye and mouth are repeatedly exposed to infectious organisms. The fact that these areas are not frequent victims of infection speaks to a complex system of immunological protection. These tissues employ not only antibodies, but also the use of small molecules with antimicrobial properties. Iodine and iodine containing compounds likely play an important role in protecting the body against infection. Through the action of various enzymes, iodine can form a number of active compounds important in fighting disease. In the presence of a peroxidase, such as lactoperoxidase and hydrogen peroxide, iodide is activated to hypoiodous acid (HOI), which is a potent antimicrobial. In addition, unincorporated iodine itself is an important disinfecting agent.

Selenium

Selenoproteins in Poultry Production GSH-Px (4 forms) Selenoprotein P Selenoprotein W

Thioredoxin reductases (3 forms)

Iodothyronine 5’ deiodinases (3 forms)

Sperm capsule Selenoprotein PH-GSH-Px

Antioxidant defense

Redox regulation of gene expression

Thyroid Metabolism

Sperm structure integrity

= Some questions remain

Diseases: Muscle dystrophy Gizzard myopathy (turkey) Exudative diathesis Pancreatic atrophy Encephalomalacia Decreased: Egg production, hatchability, chick viability, growth, immunocompetence, meat quality, feed efficiency

Poor feathering Increased cold sensitivity

Sperm motility, viability and fertilizing capacity

Metabolism of selenomethionine, selenite and selenate General body Proteins

Food

Supplements

Selenate

Selenomethionine

Selenoproteins

Selenite

Selenocysteine

Selenophosphate

GS – Se - SG

Hydrogen selenide: H2Se Methylselenol: CH3SeH Dimethylselenide: (Ch3)2Se

Breath

Trimethylselenonium: (Ch3)3Se+

Urine

Surai, Metabolism of selenomethionine, selenite and selenate (Adapted from Schrauzer, 2000; Combs, 2001; Meuillet et al., 2004)

The Metabolic Pathway of Selenium foods

SeO2 SeCys-proteins

H2O2, O2-

seryl-tRNAUGA

(GPXs, TRs, TDIs, P, W, etc.)

SeCys

H2Se

SeMet

CH3SeH GSSeH

N-AcGalNH2

GSSe-N-AcGalNH2

SeO3-2 SeO4-2

CH3SeCys

α,γ-elimination

general proteins

CH3Se-N-AcGalNH2

(CH3)2SeH (CH3)3Se+

Combs, 2001

The Differing Metabolic Pathways of Inorganic and Organic Selenium Liver gene expression by microarray analysis proved the source of the Se matters. The affected genes are involved in: 1. Nutrient metabolism. 2. Cellular growth, proliferation and immune response. 3. Cell Communication or signaling. 4. Tissue/organ development and function. Three distinct groups of genes were identified; 1. Affected by Both Inorganic Selenium and Organic Selenium Supplementation: - Up-regulated mitochondrial gene expression capacity. 2. Affected by Inorganic Selenium, only: - Down-regulated gene expression of protein involved in anti-viral capacity. 3. Affected by Organic Selenium, only: - Reduced levels of mRNA encoding proteins known to be up-regulated during oxidative stress and cancerous states.

The Metabolic Pathways for Organic Selenium and Inorganic Selenium explanation Organic Selenium: When methionine is limiting, a greater percentage of selenomethionine is incorporated nonspecifically into body proteins in place of methionine because the methionine-tRNA cannot distinguish between methionine and selenomethionine. Therefore, it is normally made bioavailable primarily due to normal recycling of body proteins. Alternatively, it can be converted through the transulfuration pathway to selenocysteine, which in turn is degraded to hydrogen selenide by the enzyme beta-lyse, thereby reentering the hydrogen selenide pool. Inorganic Selenium:

Inorganic compounds like selenite are metabolized to hydrogen selenide via selenodiglutathione and glutathione selenopersulfide. Hydrogen selenide is the precursor for supplying selenium in an active form for the synthesis of selenoproteins through the hydrogen selenide pool.

Effect of Trace Mineral Supplemental Selenium Source on Broiler Performance and Plasma Selenium Glutathione Peroxidase Study Number: 2002-SEM-1-B Solution Biosciences, Inc. Salisbury, MD

Ration Number SEM 15-1 SEM 15-2 SEM 15-3 SEM 15-4

2312 birds total.

TEST MATERIAL Sodium Selenite Selenium Yeast L-Selenomethionine ½ L-Selenomethionine½ Sodium Selenite

Added Level 0.3 ppm Se 0.3 ppm Se 0.3 ppm Se 0.3 ppm Se

Effect of Supplemental Selenium Source on Performance and Glutathione Peroxidase Levels Day 0 - 49

Sodium Selenite

Average Body Weight Gain (g) (Period)

2242.0

2249.0

2246.0

2255.0

a

a

a

a

1.860

1.838

1.862

1.851

a

a

a

a

4.902

5.686

4.902

5.686

a

a

a

a

84.337

82.918

82.915

83.625

a

a

a

a

STAT Feed Conversion Corrected STAT Mortality (%)

STAT Selenium Glutathione Peroxidase (mg/mL) STAT

Selenium L-Selenomethionine Yeast L-Selenomethionine Sodium Selenite

The Importance of Selenium during Spermatogenesis in Poultry A. Spermatozoa is a potential source of ROS activity. The reasons behind the enhanced capacity for ROS generation exhibited by defective spermatozoa, frequently appear to involve defects during spermatogenesis leading to the retention of excess residual cytoplasm in the midpiece of the spermatozoa. As spermatozoa complete their differentiation, they adopt the very unusual strategy of discarding most of their cytoplasm, just before they are released form the germinal epithelium. The small amount of residual cytoplasm that remains is concentrated in the midpiece of the cells in the vicinity of the mitochondria. Occasionally, this process is defective and excess cytoplasm is retained by the spermatozoa in the form of a “cytoplasmic droplet”. B. In chickens, a low Se diet increased the percentage of spermatozoa with bent mid-piece up to 18.7%, while dietary inclusion of sodium selenite or selenium methionine decrease this parameter by 6.2 and 0.7%, respectively. It is therefore concluded that selenium supplementation of the male diet is needed to maintain sperm membrane integrity and sperm motility during in vitro sperm manipulation during artificial insemination. The combination of vitamin E supplementation along with selenium could further improve semen quality In the following slide is a figure representing the projected effect of marginal or deficient selenium status of male fertility.

Representation of ROS Excess and Se-Deficient Effect on Spermatozoa Normal A Abnormal spermatozoon spermatozoon

Normal B spermatozoon

Head

Midpiece

Se-deficient spermatozoon

Head-Poultry (long and narrow)

Cytoplasmic droplet

Broken Midpiece

Excess cytoplasm in sperm mid-piece

Representation of normal and

Se-deficient chicken spermatozoa Tail

Excess levels of cytoplasmic Enzymes LDHC4 CK SOD G6PDH

Peroxidative damage

H2O2

NADPH

Excess ∙O2¯ production

Proposed Mechanism by which the retention of excess cytoplasm in the midpiece of the spermatozoon leads to excessive generation of ROS and the stimulation of peroxide damage. G6PDH, glucose-6-phosphate dehydrogenase; SOD, superoxide dismutase; LDHC4, testis-specific isoform of lactic acid dehydrogenase.

Representation of Protective Effect of Feeding Selenium on Spermatozoa Se in the feed

Biosynthesis of >25 selenoproteins

Testes: classic GSH-Px, PH-GSH-Px selenoprotein P, other selenoproteins

Mild Se deficiency: Se is preferably retained in the testis Progressive deficiency: morphological alterations of spermatids and spermatozoa Extreme deficiency: complete disappearance of mature germinal cells

Spermatozoa: High levels of PUFA’s (20:4n-6, 22:4n-6, 22:5n-3, 22:6n-3) + GSH-Px, PH-GSH-Px, mitochondrial capsule selenoprotein Stress conditions of sperm Manipulation (dilution, storage, deep freezing) and

ROO* = AO(vit.E) = ROOH (toxic)

free radical production H2O2+Fe=OH* ROOH + GSH-Px – ROH (nontoxic) O2+SOD=H2O2 (toxic) + GSH-Px=H2O Second line of antioxidant defense

First line of antioxidant defense Lipid peroxidation Sperm membrane damage Sperm function compromised Fertilizing capacity decreased

Magnesium

The Metabolic Significance of Magnesium 1. Each molecule of myosin (muscle protein) has an atom of magnesium in it. Muscles, therefore, have to have magnesium to work and/or grow. About 27% of the body’s magnesium is in muscle tissue, including the small muscle cells that make blood vessels contract and relax. 2. Magnesium is a necessary catalyst for all sorts of biochemical reactions. Among the enzymes that have been studied intensively, over 350 (more recent date puts the number at 500) need magnesium. For the sake of comparison, zinc is required for about 200 enzymes, copper for 20 to 30 enzymes, manganese for about 60 enzymes and selenium for about 25 enzymes. 3. Magnesium is directly necessary for enzymes that break down glucose, control the production of cholesterol, make nucleic acids (DNA & RNA), make proteins and metabolize fats. 4. Magnesium is necessary to the enzymatic control of potassium levels in the cells, including those of the cardiovascular system, thereby keeping sodium outside of the cells and having an effect on edema. 5. In addition to the 350 enzymes for which magnesium is directly necessary, it is indirectly necessary for thousands of others. An example of which would be the reaction that facilitates the formation the high energy phosphate bonds in ATP.

Physiological functions of magnesium Enzyme Functions

7 glycolytic enzymes T TCA cycle enzymes Membrane bound ATPases Kinases: creatine kinase Alkaline P’Tase 12 photosynthetic enzymes Membrane Functions Hormone-receptor binding Gating of Ca2+ channels Transmembrane flux of ions Adenylate/GMP cyclase system Ca2+ -Ca2+ release

Structural Functions

Proteins Polyribosomes Nuclei Acids Mitochondria Multienzyme Complexes, e.g., G-Proteins N-MDA receptor complex Membrane Channels Calcium antagonist Muscle contraction/relaxation Neurotransmitter release Action potential conduction in nodal tissue

Magnesium and Calcium 1. Magnesium is an important component of cell membranes. Calcium and sodium ions, for the most part, are kept outside the cell, while magnesium and potassium are kept inside the cell. If the level of magnesium within cells falls below normal, calcium and sodium move into the cell, while magnesium and potassium leak out. 2. Calcium excites nerves, whereas magnesium has a calming effect. 4. Calcium (with potassium) is necessary for muscle contraction, whereas magnesium is necessary for muscles to relax. 5. Calcium is necessary for the blood-clotting reaction, whereas magnesium helps prevent abnormal coagulation. 6. Calcium is mostly found in bones and gives them much of their hardness, whereas magnesium is in the bone matrix, or the soft structure of the bone along with protein, giving bones some flexibility and resistance to brittleness. 7. Higher than normal calcium inside a cell puts the cell in a hyperactive state. This may also lead to calcification of the cells.

Magnesium and Stress Conditions 1. Loading and transporting animals puts them under significant stress. Dietary magnesium may help counter this stress. There is data showing that ,in pigs, there can be a significant increase in survival if magnesium is supplemented prior to transportation. When the problems with cardiac arrest in animals fed PayLean® is considered, there may be some benefit to feeding supplemental magnesium relative to animal survival under transportation stress. 2. As far as oxidative stress is concerned, magnesium in necessary for the formation of glutathione, the base compound of glutathione peroxidase, the major enzyme converting H2O2 to water, thereby playing a major part in the control of oxidative cellular damage. 3. Magnesium stimulates the biosynthesis of melatonin, I am uncertain of the status of melatonin in animal diets; however, melatonin is an important antioxidant in most animal biological systems.

Additionally 1. Magnesium has also been show to increase pellet throughput when added to swine diets. It will also allow for a 10 to 15 degree pellet temperature rise. 2. Magnesium has been used to decrease tail biting in pigs.

3. Etc.

The Metabolic Significance of Magnesium The most useful method of classifying magnesium-activated enzymes is that proposed by Mahler, who divided such enzymes into five groups according to the type of reaction they catalyze: 1. 2. 3. 4. 5.

Transfer of substituted phosphoric acids. Transfer of acyl groups Hydration-dehydration of tautomerization reactions. Carbonyl addition reactions Dehyrogenations

The first group is by far the largest and it included kinases, synthetases and phosphatases. Considering the central position of phosphorylated compounds in energy metabolism, this action of magnesium alone demonstrates its importance in cellular function. Although the other four groups contain smaller numbers of enzymes, many of them are involved in the major pathways of metabolism.

Reactions of the Glycocitic Pathway Requiring Magnesium Glycogen Glucose 1-phosphate

Glucose

Mg Mg Glucose 6-phosphate Mg Fructose 6-phosphate Mg Fructose 1,6-bisphosphate Dihydroxyacetone phosphate

Glyceraldehyde 3-phosphate

1,3 Bisphosphoglycerate Fig. 1. Reactions of the glycocitic pathway requiring Mg. The enzymes activated by Mg are hexokinase, phosphoglucose isomerase, phosphofructokinase, phosphoglycerate kinase, phosphoglyceromutase, , enolase, and pyruvate kinase. In addition, phosphoglucomutase requires Mg for glycogenesis

Mg 3-Phosphoglycerate Mg 2—Phosphoglycerate Mg Phosphenolpyruvate

Mg Pyruvate

Adenine

Mg

Ribose

P

Adenine P

Ribose

ADP

P Free ATP

Adenine Ribose

Mg

Mg P

P

P

Enzyme Bound ATP

Magnesium complexes with nucleotides

P

P

Conclusions Magnesium is the natural activator of a wide range of enzymes, including many that are involved in the central pathways of cellular metabolism. It produces this action in two ways: 1. Binding with specific sites on enzymes which function as magnesium-protein complexes. This allosteric activation appears to be due to the binding of magnesium altering the conformation of the enzyme. 2. Forming part of the reactive substrate. This is the more common method of activation and complex formation with ATP is the foremost example of it. The preceding technical information was drawn from “Metal ions in biological systems’, Volume 26, published in 1990 and edited by Helmut Sigel and Astrid Sigel.

The Second Tier……. Manganese, Zinc and Copper ……. and Iron

Manganese The characteristic oxidation state of Mn in solution, in metal enzyme complexes and in metalloenzymes, is Mn2+ . Similar to Fe3+, Mn2+ has a high affinity for imidazole, in contrast to divalent cations like Zn2+ and Cu2+ that have higher affinities for thiol. Although considered rare, an experimental Mn deficiency results in a number of structural and physiological defects, including: 1) Membrane damage. 2) Altered lipoprotein synthesis. 3) Bone damage. and 4) Abnormal carbohydrate metabolism.

Mn-superoxide dismutase (MnSOD), and enzyme of which manganese is an essential component. MnSOD is found primarily in the mitochondria and is present in the enzyme in a trivalent state. Its catalytic role involves reduction, and then, re-oxidation of the metal center during successive encounters with oxygen. In addition to MnSOD, non-enzymatic forms of Mn can play a role in the destruction, as well as the formation of oxygen radicals. Low molecular weight Mn complexes inhibit lipid peroxidation in in vitro systems containing microsomes, lysozomes and cell membranes. Mn complexes apparently act as antioxidants via their ability to reduce or block the formation of OH˙, possibly through scavenging O2¯˙ or H2O2. Thus, whereas the ability of Mn to react with oxygen species is shared with that of Fe and Cu, there are fundamental differences between Mn and these two metals with respect to the mechanisms by which they react with oxygen complexes.

Manganese Dependent Enzyme Systems Category Oxido-Reductases CHOH donor

Enzyme/Substrate

CH-CH donor H2O2 acceptor

(NAD) methylene-THF dHase Peroxidase Catalase Superoxide dismutase Cholesterol-7alphs-hydroxylase

O2¯· acceptor (Other) Transferases 1-C groups CHO or CO groups Acyl groups Glycosyl groups

Alkyl, aryl groups Nitrogenous P-Containing -OH acceptor

Nucleotidyl

S-containing Hydrolases Esters carboxylate P-monoesters

Isocitrate dehydrogenase 6-P-gluconate dehydrogenase

_ Transketolase Monoacyl-glycerol-acyltransferase Glucosyl-, glycosyl Galactosyl GlcNAc NAM-, sialyl Met-adenosyl Farnesyl -

Protein kinases (Tyr,Ser) protein kinases Hexokinase P-inositol(-4-P) kinase DNA polymerase RNA polymerase Reverse transcriptase Sulfo-groups Glutathione

Mono-, di-, triglycerides Protein phosphatases P-inositol phosphatase 5’ -nucleotidase

Category P-diesters

Exp Dnase Exo Rnase Peptide Bonds

C-N bonds Linear amides

Cyclic amides Amidines Acid anhydrides P-containing C-containing Halide bonds C-X P-X Lyases C-C bonds C-O bonds P-O bonds

Isomerases Ligases (synthetases) C-O bonds CN-bonds C-C bonds P-ester bonds

Enzyme/Substrate Fructose-1,6-bisphosphotase P-Lipase C (inositide) Cyclic nucleotide phosphodiesterase Dnase (diptheria toxin) Rnase (liver) Specific proteases Metalloprotease Leu aminopeptidase Prolidase Other Aminoacylase, amidase GlcNAc-6-P deactylase Methylene-THF cyclohydrolase Arginase ATPase

Soman hydrolase PEP carboxykinase §-aminolevulonate dehydrase Adenylate cyclase Guanylate cyclase Nucleotide cyclase

AA-tRAN synthetases Glutamine synthetase Gamma-Glu-Cys synthetase Pyruvate carboxylase Vitamin K carboxylase ATP synthase

Copper Copper exhibits mono- and di-valence, and forms water-soluble cationic simple salts. Copper metalloenzymes are involved in oxidation-reduction reactions with O2 acting as an electron acceptor, and Cu is involved in the electron transfer. In tissues, Cu+ ions are oxidized to Cu2+ by peroxide, which, when bound to –SH-containing compounds, can reversibly react to form Cu+ and disulfide. Although dietary Cu deficiency is thought to be rare in humans, it is fairly common occurrence in sheep and cattle because of low forage content of copper or high molybdenum content. Four antioxidant enzymes whose activities have been reported to be reduced under conditions of Cu deficiency are: 1) Cu,Zn superoxide dismutase (CuZnSOD) - an enzyme that catalyzes the conversion of O2¯˙ to H2O2. 2) Ceruloplasmin - a Cu-containing enzyme that can act as a circulating free radical scavenger by virtue of its SOD activity and may also prevent redox cycling as a result of its ability to maintain iron in it ferric form. 3) Catalase. 4) Selenium-dependent glutathione peroxidase (GSH-Px), - which converts hydroperoxy compounds to hydroxyl compounds.

Some Copper Proteins in Humans Enzyme

Functions

Blue proteins Cytochrome-c oxidase Ceruloplasmin (ferroxidase I) Cu-Zn superoxide dismutase (SOD) Dopamine Betas-hydroxylase Diamine and monamine oxidase Lysyl oxidase Tyrosinase Chaperone proteins Chromatin scaffold proteins Clotting factor V, VIII Metallothionein Nitrous oxide reductase

Electron transfers Electron transport; reduction of O2 t H2O Iron oxidation and transport Antioxidant defense Hydroxylation of dopa in brain Removal of amines and diamines Collagen cross-linking Melanin formation Intracellular copper transport Structural integrity of nuclear material Thrombogenesis Metal sequestration Reduction of NO2¯ to NO

Zinc Zinc forms water soluble salts exhibiting a valence of +2, except at elevated pH, where it forms insoluble Zn(OH)2. Zinc readily complexes with amino acids, nucleotides, peptides and proteins, and has a high affinity for thiol [an organo-sulfur compound that contains a carbonbonded sulfhydryl (-C-SH or R-SH) group (where R represents an alkane, alkene or other carbon containing moiety)] and hydroxyl groups, and for ligands containing N as a donor. In contrast to Cu and Mn, Zn does not undergo oxidation-reduction reactions, and thus, it is not involved in electron transfer reactions. Zinc is involved in over 300 enzymes. As a result of its chelation properties, Zn serves a role in the structure and function of bio-membranes. It has been suggested that many of the pathological signs of Zn deficiency may be explained by a general membrane defect which may be related in part to excessive lipid peroxidation. There are at least three mechanisms by which Zn is directly involved in antioxidant defense. 1. The first involves its presence in the enzyme Cu,ZnSOD. Whereas the enzyme has an absolute requirement for Zn, under most conditions, there is normal activity of this enzyme, even under conditions of severe zinc deficiency. Therefore, this component of the antioxidant defense system does not appear to be susceptible to Zn deficiency. 2. Zinc also functions as an antioxidant through its interactions with specific cell surface components. In this manner, it may compete with redox active metals for membrane binding sites, thereby preventing formation of hydroxyl radicals resulting from redox cycling. 3. Zinc may also function in free radical defense through its association with the sulfur rich, low molecular weight protein, metallothionein (mt). Zn-mt can be induced by a number of conditions that increase oxidative stress, including hyperoxia, ionizing radiation and exposure to xenobiotics. It has been suggested that the Zn thiolate clusters present in protein are efficient hydroxyl radical scavengers.

Zinc, continued 4. An additional thiol compound influenced by Zn status is GSH; its synthesis is increased in the liver from Zn-deficient rats. In contrast to the lack of an effect of Zn deficiency on Cu,ZnSOD activity, the contribution that Zn ions make in terms of membrane stabilization, as well as the putative role of Zn-mt as a cellular antioxidant, is dependent on the Zn status of the individual. In fact, although Zn may not prevent Cu-induced peroxidation, it has the ability to stabilize membranes once the event has occurred. It has been shown that a Zn deficiency can result in increased lipid peroxidation in tissues and subcellular organelles, including liver mitochondria and microsomes, lung microsomes and maternal and fetal liver. As occurs in Cu deficiency, increased tissue concentrations of Fe are a common feature of Zn deficiency. This lends support to the concept that one antioxidant function of Zn is to prevent the accumulation of membrane Fe and its subsequent free radical-promoting effects. This is thought to occur through the donation of Zn by Zn-mt following its oxidation and release of thiol groups which, in turn, release Zn to compete with Fe for membrane binding sites, thereby reducing or preventing Fe redox-catalyzed lipid peroxidation.

The following slide demonstrates important biological and biochemical pathways involved in inflammation which may be influenced (direct or indirect up-or-down regulation) by copper and zinc. The inflammatory stimulus elicits the reaction of different types of both resident and circulating cells as well as the activation of some components of the complement. These cells and biomolecules cause, in turn, a cascade of events that may lead to chronicity inducing a Pathological Immune Reaction (PIR). The continuous black lines indicate the main link between the different events, while the red lines indicate secondary or indirect connections. IL-1 = interleukin 1; IL-6 = interlukin 6; COXs = cyclooxygenases; iNOS= inducible nitric oxide synthetase; LAF-1 = lymphocyte function agent 1; ICAM-1 = intercellular adhesion molecule 1; LTs = leukotrienes; PGs = prostaglandins.

R. Melanino, et al.; Copper and Zinc in the Pathophysiology and Treatment of Inflammatory Disorders, Theraputic Uses of Trace

Figure 2

Minerals, Plenum Press, NY, 1996

Inflammatory Stimuli -----------------Cocktail of Cytokines

Cu

and Other Mediators

IL 1 IL 6 Granulocytes Monocytes Macrophages

Zn

Mast Cells

Zn

HISTAMINE

Cox S

iNos

Cu

Cu

NADPH Oxidase

Cu

ARACHIDONIC ACID

B2 INTEGRIN

Zn Lipoxygenase

LTs

NO

PGs Vasodilatation and Increased Vascular Permeability

Complement

Cu Zn

Radicals

P.I.R.

Cu Zn

Proteoglycan Synthesis

Chondro-Protection Cell Adhesion ------

Tissue Repair

Migration

Cu Zn

Inflammation

-------Chemotaxis

P.I.R.

Cu

LAF 1 ICAM 1

Oxygen Free

Cu Zn

Lymphocytes Chondrocytes Endothelial Cells

Phospholipdase A2

Cu

L

Chronicity

P.I.R.

Cu Zn

PARC INSTITUTE ZINC BIOAVAILABILITY TRIAL (99-SEM-10-BB)

SEM Minerals, L.P. Zinc Sulfate Heptahydrate = Reference Standard Sample

Product

A

Blended Zinc Oxides

55.13

73.00

0.00

0.00560

0.0605

0.05

B

Waelz Process

58.42

74.50

0.00

0.01910

0.0620

0.06

C

Hydrosulfide Process Byproduct

57.93

76.00

0.65

0.00070

0.0530