the effects of selenium depletion and repletion on … · a dissertation submitted in partial...

THE EFFECTS OF SELENIUM DEPLETION AND REPLETION ON WHOLE

BLOOD SELENIUM CONCENTRATIONS AND ERYTHROCYTE GLUTATHIONE

PEROXIDASE ACTIVITY IN MODERATELY-EXERCISED HORSES

By

Kelsey Johnson Nonella

A Dissertation Submitted in Partial Fulfillment

of the Requirements for the Degree

DOCTORATE OF PHILOSOPHY

Major Subject: Systems Agriculture

West Texas A&M University

Canyon, Texas

June 2014

i

ABSTRACT

Selenium is an essential trace mineral that serves as an antioxidant, and aids in

both immune function as well as thyroid hormone metabolism. The objective of this

research was to evaluate the effects of Se depletion and repletion on whole blood Se

concentrations and erythrocyte glutathione peroxidase (RBC GSH-Px) activity. Ten

geldings received 23% of the NRC’s recommended daily Se intake during the 112-d

depletion phase. After depletion, horses were stratified by whole blood Se concentrations

and evenly divided into 2 groups of 5, and assigned to 1 of 2 treatments: 0.1 ppm organic

Se (SE1) and 0.3 ppm organic Se (SE3). During repletion, horses were fed their

respective diets for 112 d. Venous blood was collected at d 0, 28, 56, 84, and 112 of

depletion, and d 14, 28, 56, 84, 96, and 112 of repletion. Whole blood Se concentrations

and RBC GSH-Px activity were analyzed. Non-linear regression curves for whole blood

Se concentrations were developed for the depletion phase as well as both treatments

during the repletion phase. The curve of the regression equation, during the repletion

phase, were compared and were not significantly different. Whole blood Se

concentrations were compared using t-tests, and were significantly greater in horses

receiving SE3 at d 14, 28, 56, 84, 96, and 112 as compared to horses consuming SE1.

Due to the large variation in RBC GSH-Px activity, non-linear regression curves could

not be developed, and there were no significant differences between treatments within

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time throughout the repletion phase. A possible explanation for the wide variation

observed in RBC GSH-Px activity is the handling and storage of blood samples, as this

enzyme is very sensitive to temperature, especially during centrifugation. Results from

this study indicate that feeding Se above that of the NRC recommendation to previously

depleted horses may be beneficial, however never reached original values in moderately-

exercised horses.

Key words: Selenium, whole blood, glutathione peroxidase activity, depletion, repletion,

horse

iii

ACKNOWLEDGMENTS

Numerous people have helped me in my pursuit of receiving a Doctorate of

Philosophy degree. First, I would like to thank my mom and dad. They have shown me

the value a strong work ethic has, furthermore they have instilled the importance and

value a quality education has in assisting me to achieve my life goals. Throughout this

journey, they have been a constant source of support. Without them, I would not have the

bright future which lays ahead of me.

To Roger Nonella, my husband, you kept me calm and was a faithful listener

throughout this experience. You never complained about feeding for me or assisting me

during my collections. I am very thankful for your support, and that I was able to share

this experience with you.

Thank you to my committee members; Dr. Lance Baker, Dr. John Pipkin, Dr.

David Parker, Dr. Mallory Vestal, and Dr. Marty Rhoades. Dr. Baker, you helped me to

gain a deeper understanding of my research, and helped to ease my nerves when my

research did not go as anticipated. Dr. Pipkin, you have helped me grow into a better

person, people manager, and have been a consistent line of communication. Dr. Parker,

thank you for allowing me to use the CORE laboratory to prepare and store samples. Dr.

Vestal, you have helped to guide me to look at the business side. Dr. Rhoades, thank you

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for aiding in the statistical analysis of my research. This project would not have been

possible without the funding from Horse Guard, Inc. and Killgore Research Grant.

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Approved:

__________________________________________ ____________

Chair, Thesis Committee Date

__________________________________________ ____________

Member, Thesis Committee Date

__________________________________________ ____________


_________________________________________ ____________


__________________________________________ ____________


______________________________ ____________

Head, Major Department Date

______________________________ ____________

Dean, Graduate School Date

vi

TABLE OF CONTENTS

Page

ABSTRACT i

ACKNOWLEDGMENTS iii

LIST OF TABLES x

LIST OF FIGURES xi

Chapter

I. INTRODUCTION 1

II. LITERATURE REVIEW 4

Selenium Functions 4

Selenium in Soil and Forage 6

Selenium Absorption, Metabolism, and Storage 7

Selenium Absorption- Organic versus Inorganic 10

Selenium- Injectable 11

Selenium and Glutathione Peroxidase in Blood 12

Selenium Deficiency 14

Selenium Deficiency Economic Impact 15

Selenium Toxicity 16

Selenium Supplementation Environmental Impact 18

vii

Selenium in Cattle 19

Selenium in Sheep 21

Selenium in Swine 22

Selenium in Horses 23

Statement of the Problem 47

III. MATERIALS AND METHODS 48

Experimental Design 48

Diets 49

Sample Collections, Preparation, and Handling 50

Laboratory Analysis 51

Inductively-Coupled Plasma Mass Spectrometry 51

Glutathione Peroxidase Activity Assay 51

Statistical Analysis 52

IV. RESULTS AND DISCUSSION 54

Depletion Phase Whole Blood Selenium Concentrations Regression 54

Repletion Phase Whole Blood Selenium Concentrations Regression 58

Whole Blood Selenium Concentrations in Horses Consuming 0.1 and

0.3 ppm Selenium at d 0 of Repletion 63





viii









Depletion Phase Erythrocyte Glutathione Peroxidase Activity

Regression 78

Repletion Phase Erythrocyte Glutathione Peroxidase Activity

Regression 79

Erythrocyte Glutathione Peroxidase Activity in Horses Consuming

0.1 and 0.3 ppm Selenium at d 0 of Repletion 80









ix





Possible Explanation for Differences in Erythrocyte Glutathione

Peroxidase Activity between Studies 92

V. CONCLUSIONS AND IMPLICATIONS 94

LITERATURE CITED 96

APPENDIX FIGURES A, WHOLE BLOOD SELENIUM

CONCENTRATIONS GRAPHS 101

APPENDIX FIGURES B, ERYTHROCYTE GLUTATHIONE

PEROXIDASE ACTIVITY GRAPHS 105

APPENDIX TABLES A 108

x

LIST OF TABLES

Page

1. Feed Analysis for Orchard Grass Hay (DM) 50

2. Selenium Analysis for Supplements and Hay (DM) 50

3. Mean Selenium Intake (mg/kg DM) 54

A-1 Individual whole blood selenium concentrations 109

A-2 Individual erythrocyte glutathione peroxidase activity 110

A-3 Individual body weights 111

xi

LIST OF FIGURES

Page

1. Non-linear regression equation throughout 112-d selenium

depletion period (d 0, 28, 56, 84, and 112) 56

2. Non-linear regression equation of selenium depletion (means at

d 0, 28, 56, 84, and 112) and forecasted to d 250 57

3. Adjusted whole blood selenium concentrations in horses

consuming 0.1 ppm selenium at d 0, 14, 28, 56, 84, 96,

and 112 59

4. Adjusted whole blood selenium concentrations in horses

consuming 0.3 ppm selenium at d 0, 14, 28, 56, 84, 96,

and 112 60

5. Non-linear regressions over 112-d repletion of treatments (0.1

and 0.3 ppm selenium) on adjusted whole blood

selenium concentrations 62

6. Overall mean whole blood selenium concentrations at d 0 of

selenium repletion 64



xii











13. Overall mean erythrocyte glutathione peroxidase activity at d 0

of selenium repletion 81











xiii



A-1 Individual whole blood selenium concentrations throughout

selenium depletion phase 102

A-2 Individual whole blood selenium concentrations throughout

selenium repletion phase 103

A-3 Non-linear regression equations throughout 112-d selenium

depletion and 112-d selenium repletion 104

B-1 Individual erythrocyte glutathione peroxidase activity

throughout selenium depletion phase 106

B-2 Individual erythrocyte glutathione peroxidase activity

throughout selenium repletion phase 107

1

Chapter 1

INTRODUCTION

Selenium (Se) is an essential trace mineral found in varying amounts in soil, and

subsequently plants grown in that soil. Several geographical areas of the United States are

notoriously Se deficient, including the Pacific Northwest, Great Lakes Region, and

Eastern Seaboard. Therefore, horses consuming feed grown in these areas are subject to

becoming Se deficient. The primary function of Se in the body is to serve as an

antioxidant, and is a rate-limiting component of the enzyme glutathione peroxidase

(GSH-Px). Glutathione peroxidase activity is greatest in erythrocytes (Ullrey, 1987).

Selenium is also a vital component of the immune system and thyroid hormone

metabolism (Koller and Exon; 1986; Daniels, 1996; Mayer, 2009). Very few studies have

reported the effects of Se depletion in horses. The current recommended dietary intake set

by the NRC(2007) is 0.1 ppm Se. Brummer et al. (2013) reported horses receiving 0.06

ppm Se had significantly lower whole blood Se concentrations at d 84 of depletion as

compared to d 0. Whole blood Se concentrations were significantly lower at d 140 of

depletion as compared to d 84. However, there were no significant differences at d 168 or

196 as compared to d 140. Furthermore, the researchers reported significantly lower

whole blood GSH-Px activity at d 84 as compared to d 0. Whole blood GSH-Px activity

was also significantly lower at d 168 and 196 of depletion as compared to d 84.

2

Previous studies have reported conflicting results about the possible benefits of Se

supplementation above the NRC Se recommendation (0.1 ppm), particularly in

previously Se depleted horses. Brummer et al. (2013) reported horses consuming 0.3 ppm

Se had higher whole blood Se concentrations as compared to horses consuming 0.12 ppm

Se at d 154. Calamari et al. (2009) reported greater whole blood and plasma Se

concentrations in horses consuming 0.39 ppm Se as compared to horses consuming 0.18

ppm Se at d 28. Richardson et al. (2006) reported significantly greater plasma Se

concentrations in horses consuming 0.45 ppm Se as compared to horses consuming 0.15

ppm Se at d 28. Shellow et al. (1985) reported no significant differences in whole blood

Se concentrations in horses consuming 0.11 and 0.26 ppm Se in an 84-d trial. However,

the authors reported plasma Se concentrations were greater in horses consuming 0.26

ppm Se as compared to horses consuming 0.11 at d 35. Janicki et al. (2001) reported

significantly greater serum Se concentrations in mares receiving 3 mg organic Se/d as

compared to mares receiving 1 mg inorganic Se/d at d 55. Richardson et al. (2003)

reported plasma Se concentrations were significantly greater at d 28 in horses consuming

0.6 ppm Se as compared to horses consuming 0.15 ppm Se.

Previous studies have also reported conflicting results on the effects of Se

supplementation on GSH-Px activity. Brummer et al. (2013) reported horses receiving

0.3 mg Se/kg DM had significantly greater whole blood GSH-Px activity as compared to

horses consuming 0.12 mg Se/kg DM at d 154. Calamari et al. (2009) reported

significantly greater plasma GSH-Px activity in horses consuming 0.29 and 0.39 mg

Se/kg DM as compared to horses consuming 0.18 mg Se/kg DM at d 84. Richardson et

3

al. (2006) reported RBC GSH-Px activity was significantly greater in horses consuming

0.45 ppm organic Se as compared to horses receiving 0.12 ppm Se. The researchers,

however, reported no significant differences in plasma and muscle GSH-Px activity over

a 56-d trial. Shellow et al. (1985) reported no significant differences in plasma GSH-Px

activity in horses consuming 0.11, 0.16, and 0.26 ppm Se over a 12-wk trial. Richardson

et al. (2003) reported no significant differences in plasma GSH-Px activity between

horses consuming 0.15 and 0.5 ppm Se throughout a 56-d study. However, the

researchers reported horses consuming 0.6 ppm Se had significantly higher RBC GSH-Px

activity as compared to horses consuming 0.15 ppm Se at d 28.

The objective of the current study was to 1) determine the depletion rate of Se in

horses consuming a Se-deficient diet and 2) compare the effects of two different levels of

organic Se supplementations on Se repletion as indicated by whole blood Se

concentrations and erythrocyte GSH-Px activity in moderately-exercised horses.

4

Chapter II

LITERATURE REVIEW

Selenium (Se), atomic number 34, is an essential trace mineral that is found in

varying amounts in feed. It is a non-metal mineral with an atomic weight of 78.96 that

exists in two forms; inorganic species, selenate and selenite, and organic varieties,

selenomethionine and selenocysteine. The inorganic forms are present in soil, which

plants accumulate and convert to organic forms (NIH, 2013).

Selenium Functions

The primary function of Se is to serve as an antioxidant. It is a rate-limiting

component of the enzyme glutathione peroxidase (GSH-Px). Glutathione peroxidase

contains 4 g of Se atoms/mol. The greatest activity of GSH-Px occurs in

erythrocytes(RBC) and liver tissue in animals (Ullrey, 1987). Glutathione peroxidase

protects cellular membranes and organelles by inhibition and destruction of endogenous

peroxides, furthermore it works in conjunction with Vitamin E to maintain the integrity

of these membranes. The enzyme catalyzes the breakdown of hydrogen peroxide and

certain organic hydroperoxides produced by glutathione during the process of redox

cycling (Koller and Exon, 1986). Selenium also counteracts the toxicity of As, Cd, Hg,

Cu, Pb, and Ag (Koller and Exon, 1986; Charlton and Ewing, 2007).

5

There are 5 other proteins that incorporate or require Se in order to be produced.

Selenoprotein is present in striated muscle (Lescure et al., 2009). Selenoflagellin is a Se-

binding polypeptide in sperm (Selenium in Nutrition, 1983). Selenocysteine-containing

protein has been reported to be involved in the transport of Se (Reddy and Massaro,

1983). The bacterial enzymes that require Se are formate dehydrogenase and glycin

reductase, which are classified as redox enzyme systems (Reddy and Massaro, 1983).

Selenium is an essential component of selenoenzymes, nicotinic acid hydroxylase,

xanthine dehydrogenase, and a bacterial thiolase, which participate in electron transfer

processes and acts as redox catalysts (Stadtman, 1983). Other selenoproteins and

selenoamino acid transfer nucleic acids have been identified, but remain undefined

(Koller and Exon, 1986).

Selenium is a vital component of the immune system. Selenium stimulates

production of Immunoglobulin M antibody-producing cells, and enhances

Immunoglobulin G production. Immunoglobulins are glycoprotein molecules that are

produced by plasma cells in response to an immunogen and function as antibodies

(Mayer, 2009). Selenium is also involved in oxidative bursts of phagocytes. Koller and

Exon (1986) reported that neutrophils, peritoneal macrophages, and pulmonary alveolar

macrophages from Se-deficient animals had low amounts of GSH-Px activity, decreased

microcidal activity, and their ability to destroy phagocytized bacteria was compromised.

In thyroid hormone production, the enzyme Type-I Iodothyronine 5’-Deiodinase

contains Se, which converts the prohormone thyroxine to triidothyronine. Triidothyronine

affects growth and development, metabolism, body temperature and heart rate. Within 4

6

to 5 wk of Se depletion in rats, activity of Type-I Iodothyronine 5’- Deiodinase was

dramatically reduced, and the ratio of thyroxine: triidothyronine changed with an increase

in thyroxine of 50 to 100% (Daniels, 1996). The authors stated that changes in plasma

thyroid hormone status are Se specific, and occur as rapidly as the changes in GSH-Px

activity (Daniels, 1996).

Selenium in Soil and Forage

Plant uptake of Se is variable, depending on the chemical form of Se in soil, soil

acidity, the climate, and the plant species (Lewis, 1995). Selenium has similar chemical

and physical properties to S, and both share common metabolic pathways. Selenium and

S compete in biochemical processes that affect uptake throughout plant development

(Sors et al., 2005). Intensive farming with S-containing fertilizers has created many crops

that are deficient in Se (Charlton and Ewing, 2007). Rapidly growing plants and legumes

tend to be low in Se. Plants grown in poorly aerated, acidic soils, soils originated from

volcanic rock, and soils with a high content of Fe or S typically have low Se

concentrations (Aleman, 2008).

Regions of the United States that are generally extremely Se deficient are the

Pacific Northwest, Northeast, Great Lake States, Atlantic Seaboard, and Florida. The

Plains States and Southwest commonly have adequate Se in soils and plants. Around the

world, Australia, New Zealand, and China have extremely low Se content in the soil, and

consequently, forage (Koller and Exon, 1986).

Soils usually have adequate levels of Se in areas with low rainfall, where minimal

leaching of Se from the soil occurs. All but four states (DE, RI, WV, and WY) have

7

reported areas of Se deficiency. Eight states (CA, CO, ID, MT, OR, SD, UT, and WY)

have reported excess Se in certain species of plants. Selenium is more readily taken up by

plants grown in more alkaline soils (Lewis, 1995).

Three types of plants have been identified that are capable of accumulating Se.

The categories are: 1) obligate Se accumulator, 2) facultative Se accumulator plants, and

3) crop plants, alfalfa, and grasses. Crops and alfalfa normally contain non-toxic

concentrations of Se, however, if they are grown in Se-rich soils, they may contain 1 to

30 ppm Se. Obligate Se accumulator plants have an unpleasant garlic-sulfur odor, which

makes them relatively unpalatable and assists grazing animals in identifying them. Horses

and other livestock will avoid eating these plants if other feed is available. Obligate Se

accumulator plants only grow in soils high in Se. These plants are capable of

accumulating up to 10-times the amount of Se present in soil and may contain up to

10,000 ppm Se. Obligate Se accumulator plants include Milkvetches, Golden weeds,

Woody asters, Prince’s plume, Astragalus, Haploppus spp., Xylorrhiza glabriuscula, and

Stanleya pinnta. Facultative Se accumulator plants do not require Se for growth, but may

accumulate up to several hundred ppm of Se when grown in soils high in available Se.

This groups of plants includes Asters, Saltbrush, Indian paintbrush, Broomweed, Beard

tongue, Gumweed, Ironweed, Bastard toadflax, Aster spp., Machaeranthera spp. Atriplex

spp., Castilleja spp., Gutierrezia spp., Penstemon spp., Grindelia squarrosa, Sideranthus

grindelioides, and Comandra pallid (Lewis, 1995).

8

Selenium Absorption, Metabolism, and Storage

There is no known homeostatic control of Se absorption (Charlton and Ewing,

2007). Absorption takes place primarily in the duodenum of monogastrics.

Selenomethionine absorption rate in the duodenum is 98 to 100% (Charlton and Ewing,

2007; EXRX, 2013). Selenomethionine and selenocysteine are actively absorbed by the

same mechanism as the amino acid transporters for methionine and cysteine (Daniels,

1996). Absorption rates of the inorganic forms of Se vary between 30 to 100% due to

luminal factors. Selenite and selenate are passively, but rapidly, absorbed. Selenate has an

apparent absorption of 95%, compared with 62% for selenite (Daniels, 1996).

Ruminants absorb 35 to 65% of Se from forages and concentrates. Sodium

selenite is oxidized in the rumen, and then metabolized by rumen microorganisms.

Organic Se can be metabolized by rumen microorganisms, or absorbed in the small

intestine utilizing amino acid pathways (Charlton and Ewing, 2007).

Many factors affect Se absorption. Selenium absorption is higher when animals

consume a high protein diet (Daniels, 1996). Adequate dietary supplementation of

vitamins A, C, and E, and reduced glutathione result in enhanced intestinal absorption of

Se. Heavy metals, such as Pb, Fe, Hg, and Cu inhibit Se absorption via precipitation and

chelation (EXRX, 2013).

Once Se is absorbed, it is bound to a protein and transported in blood to tissues.

Plasma Se is primarily present as selenoprotein P. Selenoprotein P accounts for 60 to

70% of plasma Se and is also found in liver. Plasma selenoprotein P concentration is

directly dependent on dietary Se. Selenoprotein P in Se-deficient rats was decreased to 5

9

to 10% of that in control rats (Daniels, 1996). However, selenoprotein P declines less

rapidly than plasma GSH-Px when the exogenous Se supply is limited (Daniels, 1996;

Charlton and Ewing, 2007).

In tissues, Se is incorporated into tissue protein as selenocysteine and

selenomethionine (Charlton and Ewing, 2007). Daniels (1996) observed that albumin was

the main plasma acceptor of Se over the first 4 h post-ingestion, but by 8 h, Se was

primarily incorporated into selenoprotein P after processing by the liver. Animals can

endogenously synthesize selenocysteine from selenomethionine via the methionine

transamination and transsulfuration pathways with adequate concentrations of methionine

available, but cannot synthesize selenomethionine. Proteins such as those in skeletal

muscle, which nonspecifically incorporate exogenous and preformed selenomethionine or

selenocysteine, have been defined as Se-containing proteins. Proteins containing

endogenously synthesized selenocysteine are referred to as selenoproteins and are

metabolically active (Daniels, 1996).

Adipose tissue has very low concentrations of Se. Selenium is more commonly

associated with protein tissue. Research in steers and lambs has indicated that diets

adequate in natural Se produced liver and skeletal muscle Se concentrations that were

higher than those resulting from equal intakes of Se principally from sodium selenite.

Naturally occuring Se, such as selenomethione, produced relatively higher milk Se levels

as compared to inorganic Se compounds such as sodium selenite (Ullrey, 1987).

Selenium is stored in the kidney, liver, spleen, pancreas, and muscle. Kidneys

have the highest Se concentration followed by liver, spleen, pancreas, testes, heart,

10

skeletal muscle, lungs, and brain (Ullrey, 1987; Stowe and Herdt, 1992). Normal liver Se

concentrations range between 1.2 and 2.0 µg/g of dry weight for all species regardless of

age (Stowe and Herdt, 1992). However, skeletal muscle is the major site of Se storage,

accounting for approximately 28 to 46% of the total Se pool (http://ods.od.nih.gov,

2013).

Selenium homeostasis is primarily regulated by excretion. The primary routes of

excretion for monogastrics are urine and feces, and when toxic levels are consumed

excretion also occurs via lungs through exhalation. In ruminants, unabsorbed dietary Se is

excreted through the feces, and injected Se is excreted through urine. Se retention was

reported to be influenced by animals’ Se status, and the amount and chemical form of Se

fed (Charlton et al., 2007). Much of tissue Se is labile, and following transition from

seleniferous diets to low Se diets, losses from the body are rapid initially and then

decrease (Ullrey, 1987).

Selenium Absorption- Organic versus Inorganic

Plant forms of Se are the same as organic forms in yeast, which is the form that

horses naturally consume. The inorganic forms of Se are a by-product of Cu mining.

Organic Se, predominantly selenoamino acids and related compounds, are more easily

digested, metabolized, and retained in tissues. Organic Se is much safer to feed to

livestock than inorganic Se because selenoamino acids are absorbed from the gut via

amino acid pathways, which aids in limiting excessive absorption of Se. Selenite Se is

passively absorbed, which allows rapid and unregulated uptake of possibly toxic levels of

11

Se. Organic Se is also safer to handle because it is not absorbed through human skin like

sodium selenite (Equine Nutrition, 2005).

In order for Se to be incorporated into selenoproteins, dietary sources of Se must

be inserted into cysteine where Se replaces the thiol (-SH) side chain, thus forming the

amino acid residue selenocysteine. Inorganic species of Se (selenite and selenate) must

first be reduced to selenide before being incorporated into selenocysteine residues.

Sodium selenite is the most common inorganic form of Se supplemented to horses.

Apparent absorption of selenite in mature horses was reported to be 51.1% (Pagan et al.,

2007). Selenomethionine is the most common organic form of Se fed to horses, and is

most prevalent in plants and yeast. Apparent absorption of selenomethionine was shown

to be 57.3% in horses (Pagan et al, 2007). Selenomethionine is actively transferred

through the intestinal membrane and can replace methionine during protein synthesis.

Selenium is not catalytically active in selenomethionine form and must be converted to

selenocysteine. Dietary sodium selenite is more rapidly incorporated into GSH-Px in

serum than selenomethionine, but is not stored in tissues as much as selenomethionine

(White, 2010).

Selenium- Injectable

Injectable Se products administered immediately before competition have been

gaining in popularity because of their possible performance-enhancing qualities in

equine. In April 2009, 21 polo ponies in South Florida died after receiving an injectable

Se supplement containing an acutely toxic concentration of Se. The compounding

pharmacy responsible for creating the supplement miscalculated the amount of Se to be

12

added to the injection (Desta et al., 2011). This example highlights the narrow margin

between the Se requirement of animals and Se toxicity, especially with injectable Se

(White, 2010).

In addition, adverse responses to injectable Se/vitamin E products have been

observed in several species and animal owners should be advised of the potential fatal

effects of these products, even when used at recommended doses. The response is an

immediate, usually fatal, anaphylactic reaction (Stowe, 1998). The reaction is not to the

Se or vitamin E in the product but apparently to an emulsifying agent or preservative

present in the product. None of these untoward reactions is observed from oral

administration of Se at appropriate rates (Stowe, 1998).

Selenium and Glutathione Peroxidase in Blood

Whole blood Se is a good measure of Se intake because it represents both serum

and RBC Se, and appears to be a more preferable indicator of Se status than serum (NRC,

2007). However, whole blood Se responds more slowly than serum or plasma to changes

in dietary Se intake because a majority of the glutathione peroxidase in whole blood is

incorporated into the RBC at the time of erythropoiesis, and changes very little over the

life of the cell. A measurable response in whole blood Se to a Se supplement, therefore,

requires a time span equal to the average life span of RBC. In cattle, the life span of a

RBC is about 90 to 120 d. (Stowe and Herdt, 1992). Carter et al. (1974) reported the

lifespan of erythrocytes in light horses to be 145 to 165 d. The whole blood Se: serum Se

ratio is approximately 1:1 in swine, 1.4:1 to 1.5:1 in horses, 2.5:1 in dairy cattle, and 4:1

in sheep, particularly neonates (Stowe and Herdt, 1992). These ratios would initially

13

narrow after an increase in oral Se intake and initially widen on cessation of Se

supplementation. In swine, the correlation coefficients of Se concentrations of these

tissues related to plasma Se are 0.95, 0.91, and 0.71 for skeletal muscle, liver, and kidney,

respectively, on a wet weight basis (Stowe and Herdt, 1992).

Whole blood GSH-Px activities are consistently measurable. Complete GSH-Px

activity response to Se supplementation requires about 80 to 90 d, equal to the life span

of equine erythrocytes, as Se is only incorporated into RBC during erythropoiesis. Whole

blood GSH-Px concentrations range from 40 to 160 units of enzyme activity(mU)/mg

(hemoglobin) Hb in horses (Stowe, 1998). There is a high correlation between

erythrocyte GSH-Px activity and Se concentrations in whole blood of humans, cattle,

sheep, horses, and swine with a low Se status; however, these correlations became much

weaker as Se status increased (Ullrey, 1987). Whole blood has a 10 to 50% higher Se

concentration due to the significantly higher concentration of Se in erythrocytes than in

plasma. In sheep blood, GSH-Px activity in erythrocytes was 99-times that of plasma

(Ullrey, 1987). In erythrocytes, plasma GSH-Px activity ratio in cattle is 49:1, and 26:1 in

swine (Ullrey, 1987).

Animals are considered to be sub-clinically deficient when whole blood Se

concentration and GSH-Px activity is less than 0.05 ppm and 30 mU/mg hemoglobin,

respectively. Selenium and GSH-Px statuses are considered marginal between 0.05 to 0.1

ppm and 30 to 60 mU/mg hemoglobin. Blood Se concentrations and GSH-Px activity

greater than 0.1 ppm and 60 mU/mg hemoglobin, respectively, are considered adequate

(Koller and Exon, 1986).

14

Selenium Deficiency

Signs and symptoms of Se deficiency are similar for both animals and humans.

Severe Se deficiency is characterized by cardiomyopathy. Moderate deficiency is

characterized in less severe, myodegenerative symptoms such as muscular weakness and

pain. Symptoms range from the well-recognized, ominous, severe condition of nutritional

muscular dystrophy i.e., “white muscle disease” to numerous, less explicit conditions

often referred to as Se-associated or Se-responsive diseases. Selenium-associated diseases

are characterized by muscular weakness, unthriftness, reduced weight gain, diarrhea,

stillbirths, abortions, and diminished fertility (Koller and Exon, 1986; Charlton and

Ewing, 2007).

White muscle disease is a myodegenerative disorder most commonly associated

with neonates. The disease occurs most often in lambs, but has also been observed in

calves and horses. Young animals may die suddenly due to myocardial dystrophy.

Subacute symptoms are stiffness, weakness, and trembling of the limbs, frequently

followed by the inability to stand, and swollen muscles that feel hard to the touch.

Affected animals also exhibit dyspnea and labored breathing from involvement of

diaphragm and intercostal muscles (Koller and Exon, 1986; Charlton and Ewing, 2007).

In horses, Se-deficiency symptoms are more ambiguous than in other livestock

species. Selenium-deficient horses can experience myopathies such as myositis,

polymyositis, and azoturia. Infertility is also commonly observed in deficient horses.

15

Finally, muscular weakness in foals and reduced performance during exercise are

common symptoms of Se deficiency (Koller and Exon, 1986; Charlton and Ewing, 2007).

A muscular disorder associated with Se/vitamin E deficiency in horses is a non-

exertional myopathy with rhabdomyolysis. It is a peracute to subacute myodegenerative

disease of cardiac and skeletal muscle caused by a dietary deficiency of Se, and to a

lesser extent vitamin E (Aleman, 2008). This disease occurs primarily in young growing

foals, but has also occurred in older horses. Peracute clinical signs in foals include

recumbency, tachypnea, myalgia, arrhythmias, and sudden death. Subacute signs include

severe weakness, inability to stand, muscle fasciculations, firm muscles on palpation,

stiffness, stilted gait, myalgia, lethargy, dysphagia, trismus, ptyalism, and a weak suckle

reflex. Physiological alterations in horses with low whole-blood Se and GSH-Px activity

include high serum creatine kinase and aspartate aminotransferase activities,

hyperprotienemia, azotemia, hyponatremia, hypochloremia, hyperkalemia,

hyperphosphatemia, respiratory acidosis, and myoglobinuria (Aleman, 2008). Also,

muscles are pale with white streaks, representing coagulative necrosis and edema. The

muscles most affected are the myocardium, thoracic, pelvic and cervical muscles,

diaphragm, tongue, pharynx, intercostals and masticatory muscles (Aleman, 2008).

Clinical manifestations of many of these disorders require contributory factors, such as

stress, to precipitate symptoms (Koller and Exon, 1986).

Selenium Deficiency Economic Impact

Selenium deficiency caused enormous yearly economic loss on producers before

Se supplementation was permitted (Koller and Exon, 1986). Prior to the allowance of Se

16

supplementation, 15 to 20% mortality, and at least 25% morbidity was observed in

growing pigs of swine herds. In addition, reproductive efficiency was lower and

resistance to environmental stress and infectious disease was diminished. Comparable

death losses and declines in productivity were observed in poultry and other livestock

species (Ullrey, 1992).

The USDA approved Se supplementation at a rate of 0.1 ppm in 1974. Prior to

this, the inability to supplement deficient poultry and swine caused U.S. producers annual

losses of over $82,000,000 (Ullrey, 1992). Dietary supplementation at 0.1 ppm was

approved for beef cattle, dairy cattle, and all ages and gender of sheep in 1979. Prior to

this approval, estimated annual loss for U.S. producers of beef cattle, dairy cattle, and

sheep was approximately $545,000,000 in 1976 (Ullrey, 1992). Subsequent research

provided evidence that additional Se could be beneficial. In 1987, a maximum level of

supplemental Se from 0.1 to 0.3 ppm in complete feeds for all major food-producing

animals was approved by the FDA (Ullrey, 1992).

Selenium Toxicity

Selenium was first identified as a toxic element that induced hair loss, lameness,

hoof sloughing, and death in grazing livestock in SD and WY in 1934 (Ullrey, 1992).

Marco Polo and T.C. Madison observed similar signs in horses in China in the 13th

century, and at Fort Randall, NE Territory in 1860, respectively. T.C. Madison called the

toxicity “alkali disease” (Ullrey, 1992). Toxic concentrations of Se inhibit cellular

enzyme oxidation-reduction reactions, especially those involving sulfate or S-containing

amino acids methionine and cysteine, which affect cell division and growth. Hoof and

17

hair are especially susceptible to the effects on cell division (Lewis, 1995). Selenium

toxicity is usually associated with incorrect feed levels or eating Se-accumulating plants.

Feeds containing more than 5 ppm Se are considered Se toxic. The Maximum Dietary

Tolerable Level has been established at 2 mg/kg DM (Charlton and Ewing, 2007).

Acute signs of Se toxicity include garlicky breath, vomiting, dyspnea, titanic

spasms, apparent blindness, head pressing, perspiration, abdominal pain, colic, diarrhea,

increased heart and respiration rates, and death from respiratory failure (Koller and Exon,

1986; NRC, 1989). Death due to pulmonary congestion and edema occurs from acute Se

toxicity with 25 to 50 mg/kg (Lewis, 1995). Chronic toxicity occurs when an animal

consumes 5 mg/kg or more of Se (Koller and Exon, 1986). Chronic poisoning symptoms

are abnormal hoof and hair growth, alopecia (especially mane and tail), fetal

abnormalities, and cracking of hooves around the coronary band (NRC, 1989; Lewis,

1995).

In horses, chronic Se toxicity was reported in 25 horses used in a feedlot in NE

(Stowe and Herdt, 1992). The horses developed hair loss and lesions around the coronary

band. One horse had 928 ng Se/mL in serum as compared to an expected normal range of

140 to 160 ng Se/mL. Feed analysis determined that these horses were fed hay

containing 20 ppm Se for more than 3 wk. After 2 wk of consuming hay with non-toxic

Se concentrations, mean serum Se was 525 ng Se/mL, and 6 wk after the diet change,

mean Se serum was 285 ng Se/mL (Stowe and Herdt, 1992).

18

Selenium Supplementation Environmental Impact

Some environmental groups have raised concerns about the impact that Se

supplementation may have on the environment. For example, the Kesterson Reservoir

was essentially a wastewater dump that received irrigation drainage water from the San

Luis United of the United States of Reclamation’s Central Valley Project in western

Fresno County (Ullrey, 1992). Selenium was proposed as a cause of death and

deformities in aquatic birds and other organisms. Selenium, from seleniferous marine

rocks of Oligocene, may have been one of the factors involved. However, there is no

evidence of undesirable amounts of Se entering this ecosystem from the legal use of Se

supplements in animal diets (Ullrey, 1992). No significant differences in upstream and

downstream contents of Se in water, stream sediment, algae, invertebrates, and fish was

observed at ranches on which Se supplementation of beef cattle had been practiced for 3

to 8 yr (Oldfield, 1998).

Primary domestic production of Se in 1989 was about 250 metric tons and 450

metric tons imported, of which 40% was used for electronic and photocopier components,

20% glass manufacturing, 20% chemicals and pigments, and 20% other applications.

Less than 6.8% of 47.5 metric tons was used for supplementing animal diets. Fuel

combustion, refuse combustion, metal mining and refining, and industrial production

were identified as anthropogenic contributions of 4,670 metric tons (Ullrey, 1992). If all

the Se incorporated into animal feeds were to enter the environment, it would account for

less than 0.5% of the Se that originated from natural and other identified anthropogenic

sources. Unabsorbed inorganic Se in animal feces is largely insoluble elemental Se and

19

metal selenides and urinary trimethyl selenonium is poorly available for absorption by

wildlife and aquatic life. Therefore, the environmental threat from legal use of Se

supplementation seems very small (Ullrey, 1992).

Selenium in Cattle

Podoll et al. (1992) used 18 lactating Holstein cows, split into 2 treatments; 0.3

ppm sodium selenite and 0.3 ppm sodium selenate to determine if sodium selenate had

superior bioavailability to Na selenite as a source of supplemented Se. Serum was

collected on d 0, 3, 7, 10, 14, 28, 42, and 49 for Se and GSH-Px assays. The authors

reported serum Se concentrations rose significantly during the study in cattle consuming

both treatments. Response to supplemental Se was cubic. Sodium selenate

supplementation produced significantly greater serum Se concentrations than selenite.

Serum GSH-Px activities were unaffected by the form of Se, but were significantly

different over time (Podoll et al., 1992).

In order to determine the response of calves fed low-Se diets, then supplemented

with either Se-enriched yeast or inorganic Se, Nicholson et al. (1991a) fed 50 crossbred

beef calves (6 to 7 mo of age) and 20 yearling Holstein heifers. There were 5 treatments;

Control (no supplemental Se or yeast), Inorganic Se (Sodium selenite to supply 1 mg

Se/kg of supplement), organic Se (Alkosel yeast to supply 1 mg Se/kg of supplement),

live yeast, and autoclaved yeast (commercial yeast culture was autoclaved for 8 min at 15

psi). The experimental period was 112 d. Blood samples were collected at 4 wk intervals

and whole blood Se and GSH-Px activity was measured. The authors reported animals

fed organic Se supplement had significantly higher blood Se concentrations than those

20

not receiving supplemental Se at d 28. Differences continued to be observed for the

duration of the trial. By d 84, whole blood Se concentrations for animals fed organic Se

were numerically higher as compared to those fed inorganic Se, and the differences

became significant by d 112. There were no significant differences in whole blood Se

concentrations among the groups that did not receive supplemental Se. Whole blood

GSH-Px activity was not significantly different at d 28 among treatments, but became

significantly different at d 56 and continued remained significantly different throughout

the remainder of the trial. Differences in blood GSH-Px activities due to 2 sources of Se

became significant at d 112. Whole blood Se and GSH-Px values for cattle fed inorganic

Se appeared to plateau between d 84 and 112, while those fed organic Se appeared

continued to increase (Nicholson et al., 1991a).

Nicholson et al. (1991b) used 48 growing beef cattle and 20 yearling Holstein

heifers to compare rates of change in Se concentrations of whole blood or blood plasma

due to altered dietary Se source. Animals received 2 kg concentrate with or without

addition of Se enriched yeast to supply 1 ug Se/d. Blood samples were collected for Se

analysis at approximately 28-d intervals over a 163-d period. The authors reported

increased Se levels in whole blood over 8 wk even though levels in all animals were

within the normal range at the start of the experiment. Increases were greater for animals

that received Se-enriched yeast in their supplement than for the non-supplemented

animals, but the slopes of the linear regressions did not differ significantly. When animals

were changed to low Se diets, there was a significant decline in whole blood Se for those

animals not fed Se-enriched yeast in their concentrate in the slope of the regression line

21

over the final 16 wk of the trial for whole blood concentrations. However, this significant

decline was not observed in plasma. The authors concluded that changes in whole blood

Se concentrations are a more accurate measure than plasma Se concentrations of the

adequacy of current Se intake as the magnitude of change was greater and values did not

plateau at as low a level of intake (Nicholson et al., 1991b)

Selenium in Sheep

In sheep, Wright and Bell (1966) fed 10 wethers 0.35 ppm Se 2 wk prior to, and

during the experimental period to determine Se retention. Five wethers were given a

single oral dose via a gelatin capsule, and 5 were given a single intravenous dose of

radioactively-labeled selenium. The authors observed the retention of Se after 120 hr was

29% when radioactively-labeled selenium was administered in a single oral dose.

Retention of intravenous dose of Se was 70% after 120 hr with the major route of

excretion via urine (Wright and Bell, 1966)

Podoll et al. (1992) fed 20 crossbred wethers 1 of 2 treatments; 0.3 ppm sodium

selenite and 0.3 ppm sodium selenate to determine bioavailability of each Se source.

Serum was collected on d 0, 3, 7, 10, 14, 28, 42, and 49 for Se and GSH-Px assays. The

authors observed serum Se concentrations rose significantly during the study. The

response of wethers to supplemental Se was quadratic. However, there were no

differences observed in serum Se due to treatment. Serum GSH-Px activities were

unaffected by the form of Se but were significantly different over time (Podoll et al,

1992).

22

Selenium in Swine

Wright and Bell (1966) fed 10 barrows 0.5 ppm Se daily for 2 wk prior to and

during an experimental period, consisting of a 4-d preliminary and a 5-d collection

period. Five barrows were given a single oral dose via stomach tube, and 5 were given a

single intravenous dose of radioactively-labeled selenium. Retention of oral Se after 120

hr was 77%. Retention of intravenous dose of Se was 70% after 120 hr with the major

route of excretion via urine (Wright and Bell, 1966).

Chavez (1979) weaned 16 piglets at 14 d to evaluate the biodynamic relationship

between blood Se concentrations and the activity of GSH-Px in the plasma, and Se

concentration changes in different body tissues during Se depletion and repletion. Piglets

were randomly assigned into 2 dietary treatments for 4 wk: basal diet containing 0.02

ppm Se, or basal diet supplemented with 0.1 ppm Se as Na selenite. After 4 wk, half of

the piglets from each dietary regime were changed over to the other diet for 5 wk. The

change in diet represented the depletion or repletion period for piglets fed the previous

respective dietary treatment. Blood samples were collected at weaning, and weekly

throughout the trial. The authors reported after 1 wk of receiving the experimental diets,

there was a significant difference in blood Se concentrations between the 2 groups of

piglets. At wk 4, piglets receiving Se supplement had 132 ug Se/L as compared to 27 ug

Se/L in non-supplemented piglets. Piglets receiving Se supplement had increased blood

Se concentration (a maximum of 165 ug/L in wk 9), although no significant difference

was observed during the last 3 wk of the trial. Blood Se concentration of piglets

continuously fed the Se deficient diet decreased to an average minimum value of

23

approximately 17 ug/L after 8 wk, with significant variation observed during the last 4

wk of the trial. In piglets changed from a Se deficient to Se supplemented diet, blood Se

concentration increased steadily for 5 wk, although a much faster repletion rate took

place during the first 2 wk after the change in diet. Piglets receiving Se supplementation

during the entire trial had a significant increase in plasma GSH-Px activity, while piglets

fed the Se deficient diet during the entire trial had a significant decrease. Piglets changing

from Se supplementation to a Se deficient diet had a significant decrease in plasma GSH-

Px activity during wk 1 of depletion, and this activity continued to decrease thereafter,

but at a slower rate. Piglets changed from a Se-deficient diet to Se supplementation at wk

4 had a significant and steady increase in GSH-Px activity for 4 wk after the change.

Further, this activity peaked at a value higher than that observed in the plasma of control

animals receiving Se supplementation continuously. The repletion rate of blood Se was

about 12% faster in piglets changed to supplemental Se treatment as compared to the

depletion rate of piglets changed to the basal diet (Chavez, 1979).

Selenium in Horses

Horses, zoo animals, llamas, and other pets have never been included in the FDA

regulations on Se supplementation. However, reference values for Se in mature horses

have been established; serum between 130 to 160 ng Se/mL, whole blood between 182 to

240 ng Se/mL, and liver between 1.2 to 2.0 ug Se/g DM (Stowe, 1998). Maximum

tolerable concentration of dietary Se for horses is reported to be about 2 ppm (Stowe,

1998). Therefore, a considerable margin of safety exists between the practiced 0.1 to 0.3

ppm rate of supplementation and maximum tolerable level (Stowe, 1998).

24

Historically, inorganic Se sources have been used in equine feeds, but the margin

of error for inorganic Se supplementation is narrow, and efficacy has been questioned. A

growing body of research suggests organic Se sources enhance Se incorporation into

tissues, both at rest and during exercise (Dunnett and Dunnett, 2008). Further, organic Se

supplementation has been observed to increase Se status, enzyme activities, antioxidant

capacity and immune function in mature horses and foals. Dietary organic Se appears to

cause a greater relative increase in plasma Se over 28 d as compared to selenite, although

comparative effects were similar over 56 d for skeletal muscle Se and plasma GSH-Px

activity. Dietary organic Se also produced a greater numerical, but statistically

insignificant, increase in plasma GSH-Px activity than selenite during supplementation to

horses over 112 d. Post-supplementation decline in plasma GSH-Px activity was also

reduced in horses receiving organic Se. Data from other studies have indicated that pre-

and post-partum organic Se supplementation in mares has subsequent benefits in the foals

through improved Se status (Dunnett and Dunnett, 2008).

The Se requirement for sedentary horses was estimated at 0.1 mg/kg of diet by

Stowe in 1967. Exercise increases oxidative metabolism markedly, which results in

mobilization of tissue Se to meet increased antioxidant demand, explaining why

performance horses have greater Se requirements than non-athletes (Equine Nutrition,

2005). Unlike other livestock species, the FDA only makes dietary recommendation for

Se in equine feeds. In horses, nutritional myopathy involving skeletal and cardiac

muscles is associated with GSH-Px values lower than 25 mU/mg and serum Se values

lower than 60 ng/mL. Selenium deficiency results in weakness, impaired locomotion,

25

difficulty in suckling and swallowing, respiratory distress, and impaired cardiac function.

In deficient horses, serum concentrations of creatine kinase, aspartate aminotransferase,

K, aspartic-pyruvic transaminase and gamma-glutamyltransferase are increased (NRC,

1989). Serum Se in foals from Se-adequate mares is typically much lower than their

dams, and ranges from 70 to 80 ng/mL. Serum Se values lower than 65 ng/mL are

indicative of deficiency (NRC, 1989).

Stowe (1967) obtained 12 orphaned foals initially fed a commercial milk replacer.

Foals were used to evaluate the effect of Se on growth rate before clinical evidence of

deficiency occurs. Half of the foals were fed a semi-purified diet, and other half were fed

semi-purified diet supplemented with 2 ppm Se in the form of Na selenite. The author

reported a tendency for the Se-supplemented foals to gain more rapidly than the Se-

deficient foals (Stowe, 1967).

Carmel et al. (1989) surveyed a randomly selected horse population from 4

contiguous counties in northern MD to determine the Se status of resident horses. From

the MD horse population, 203 horses from 74 farms were sampled from January through

May, 1988. Information on signalment, duration of residence, use, housing, medical

history, and feeding program was collected. Whole blood Se concentrations greater than

or equal to 0.1 ppm were considered adequate. The authors reported average whole blood

Se concentrations were 0.137 ppm, and ranged from 0.05 to 0.266 ppm. Of the horses

sampled, 18.7% were considered deficient. There was a significant negative correlation

observed between whole blood Se concentration and amount of time horses had access to

26

pastures. Horses used daily and those fed daily supplement were significantly more likely

to have adequate Se concentrations (Carmel et al., 1989).

Ludvikova et al. (2005) collected blood samples from 159 horses from 35

different farms to determine the relationship between Se concentration and activity of

GSH-Px in whole blood of horses, reference ranges for the activity of GSH-Px, and to

evaluate Se status of horses in the Czech Republic. The authors observed a highly

significant linear relationship between Se concentration and GSH-Px activity. Whole

blood Se concentrations of 75 ug/mL were considered the threshold of Se deficiency.

There was a high prevalence of selenium deficiency in horses examined. Selenium status

and GSH-Px activity was considered deficient in 47 and 48% of horses examined,

respectively (Ludvikova et al., 2005).

Blackmore et al. (1982) measured Se concentrations and GSH-Px activity in 84

Thoroughbreds to assess the relationship between Se status, and muscle and hepatic

disorders. Whole blood was collected and analyzed for Se, and any muscle or hepatic

disorders were assessed. Researchers reported a significant linear (r = 0.843) and

quadratic (r = 0.976) relationship between whole blood Se and RBC GSH-Px activity

(Blackmore et al, 1982).

Knight and Tyznik (1990) evaluated the effects of supplemental Se on equine

humoral antibody production. The authors utilized 15 Shetland ponies; five 2-yr old, four

3-yr old, and 6 yearlings. During the depletion phase, 2 and 3-yr old ponies were fed a

low-Se diet for 1 yr, and yearlings were fed similarly for 9 mo. During the depletion

period, the average GSH-Px activity decreased from 150 mU/mg hemoglobin to 20

27

mU/mg Hb. During the 7-wk repletion period, horses were assigned to 1 of 2 treatments;

low (0.02 ppm), or high (0.22 ppm). The authors stated Se supplementation had a positive

effect on the immune response. Serum Immunoglobulin G concentrations of ponies

receiving Se-supplementation were significantly higher as compared to those receiving

no Se. Older ponies had significantly higher serum Immunoglobulin G concentrations

than did yearlings. A significant interaction between Se and time was observed for serum

Immunoglobulin G concentration and hemagglutination titers. Serum Immunoglobulin G

concentrations were significantly higher during wk 2, 3, 4, and 5 in Se-supplemented

ponies. Hemagglutination titers during wk 2, 3, 4, and 5 also were significantly greater in

ponies receiving supplemental Se. Horses consuming Se supplementation had

significantly higher whole blood Se concentrations and glutathione peroxidase activity.

Whole blood Se concentrations and glutathione peroxidase activities significant increased

during the 6-wk trial in supplemented ponies. Selenium-supplemented ponies had

significant higher whole blood Se concentrations during wk 4, 5, and 6 and glutathione

peroxidase activities during wk 6. A significant positive correlation (r = 0.79) between

whole blood Se and glutathione peroxidase activity within treatment was observed

(Knight and Tyznik, 1990).

Shelle et al. (1985) conducted a study using 8 Arabian mares in a 2 x 2 double

split-plot design with repeated measures to determine the effects of conditioning,

exercise, and daily Se supplementation. Treatments consisted of 2 levels of dietary Se, no

added Se or 2.5 mg added Se/d. Exercise treatments were non-conditioned, conditioned 6

d per wk for 45 d, or conditioned for 3 d followed by 2 d stall rest for 45 d. Blood

28

samples were taken before, during and after exercise. The authors reported plasma Se

concentrations significantly increased with Se supplementation above pre-feeding levels.

Horses consuming the basal ration had a significant decline in plasma Se over the length

of the trial. Plasma Se concentrations were significantly elevated during exercise as

compared to 1 hr post-exercise. The authors hypothesized increased plasma Se

concentrations during exercise resulted from changes in plasma volume rather than

mobilization of Se from body stores. Mean plasma GSH-Px activities were 5.3 and 7.7

mU/mg of plasma protein and significantly different in non-supplemented and

supplemented mares, respectively. Furthermore, the authors stated that erythrocyte GSH-

Px activities increased as a result of conditioning. Selenium supplementation appeared to

augment the effect of conditioning and resulted in significant treatment by conditioning

interaction. Glutathione peroxidase activity in whole blood was significantly elevated

during exercise (Shelle et al., 1985).

Brummer et al. (2009) used 24 horses to establish the correlation between Se

status, as measured by serum Se concentrations, and GSH-Px activity, along with several

immune-related variables. Sixteen horses received no dietary supplementation except for

access to a salt block, while 8 horses were supplemented with a commercial grain-based

concentrate containing at least 0.3 ppm Se. Horses were fed their respective diets for 4

mo prior to blood sampling. Blood was drawn over a period of 6 wk. Each horse was

sampled once. Authors reported serum Se concentrations ranged from 69 to 193 ng/mL.

Mean serum Se concentrations were significantly different between supplemented (165

ng/mL) and unsupplemented (91 ng/mL) horses. A positive correlation of medium

29

strength was observed between serum Se and whole blood GSH-Px activity (r = 0.710).

A weak correlation was observed for serum Se and IL-10 gene expression (r = 0.419). A

trend was observed for a weak correlation between serum Se and serum GSH-Px (r = -

0.0359). There was also a trend for weak correlations between serum Se and tumor

necrosis factor expression (r = 0.381), and serum Se and tumor necrosis factor production

(r = 0.446; Brummer et al., 2009).

Chiaradia et al. (1998) studied the possible relationships between physical

exercise, lipid peroxidation and muscle fiber damage in trained horses. Researchers fed

ten 3-yr old Maremmana stallions a minimum of 12 mg Se and 1000 IU of vitamin E/d.

Stallions underwent physical training for 3 mo, 30 min/d, 6 d/wk, and intensity gradually

increased. At the end of the trial, horses performed an exercise test consisting of an 8 min

warm-up period followed by two 200-m gallops. Blood samples were collected before

exercise, immediately after warm-up, after the gallops, and 18-hr post exercise. Total

plasma glutathione, reduced glutathione and glutathione disulphide were measured.

Results indicated that the pattern of glutathione content in the plasma was similar before

exercise and immediately after warm-up, increased after the gallops, and decreased to

pre-exercise concentrations 18-hr post exercise. The authors stated that after an oxidative

stress, glutathione is released in the blood. The oxidized form of glutathione is transferred

from the cells to the liver to be reduced, and the reduced form of glutathione is then

released by the liver to support increased requirement of cells for this substrate, which is

necessary for the activity of GSH-Px (Chiaradia et al., 1998).

30

In a companion study to Chiaradia et al. (1998), Avellini et al. (1999) sought a

better understanding of the effect of dietary supplements and a 70-d training period on the

peroxidation phenomena induced by rigorous programmed physical exercise trials of

increasing intensity. The authors reported the activity of GSH-Px was significantly lower

at the beginning of the trial as compared to d 70. No significant modifications in enzyme

activity were observed after physical exercise. Glutathione peroxidase activity

significantly increased over the 70-d period of training and diet supplements. The authors

concluded training and diet supplements increased antioxidant defenses in extracellular

fluids and blood cells of the horses (Avellini et al., 1999).

Greiwe-Crandell et al. (1993) split 45 pregnant Thoroughbred mares into 3 groups

to determine mineral status of mares and foals during Se depletion. Mares were divided

into the following treatment groups; 15 fed mixed grass/legume pasture and

supplemented with hay only, 15 fed similar pasture and supplemented with hay plus 3.2

kg/d of a concentrate, and 15 dry-lotted and fed 2-yr old mixed grass hay and 4.6 kg/d of

concentrate. After foaling, weanlings remained on the same regimens as their dams. The

concentrate contained 0.6 mg/kg Se, and the pastures and hay contained 0.08 mg/kg Se.

The authors reported whole blood Se concentrations of horses fed pasture and hay only

were significantly lower than horses fed 3.2 kg/d of concentrate, and horses fed 4.6

concentrate. Whole blood Se concentrations tended to be different in November between

horses fed 3.2 and 4.6 kg/d concentrate, however, there was no difference between the 2

groups in January (Greiwe-Crandell et al., 1993).

31

Karren et al. (2010) used 28 pregnant Quarter Horse mares in a 2 x 2 factorial,

randomized complete block design to investigate the maternal plane of nutrition and the

role of Se-yeast on muscle Se concentration, plasma GSH-Px activity, and colostrum Se

concentration in mares and their foals. There were 4 treatments. Seven mares were

allowed access to pasture and received no Se supplementation, receiving a total of 0.19

mg Se/kg DM. Eight mares were allowed access to pasture and Se supplementation,

receiving 0.49 mg Se/kg DM. Five mares fed pasture and grain with no Se

supplementation receiving 0.35 mg Se/kg DM. Eight mares were fed pasture and grain

with Se supplementation receiving 0.65 mg Se/kg DM. The treatments were initiated 45 d

prior to third trimester. Selenomethionine supplementation was initiated at the beginning

of the third trimester (approximately 110 d before estimated foaling date). Blood samples

were collected every 14 d until parturition. Foal blood samples were taken beginning at

birth and every 14 d until 56 d of age. Colostrum samples were obtained after parturition

and before nursing. Mare muscle biopsies were collected every 28 d until parturition.

Foal muscle biopsies were collected at birth and on d 28 and 56. The authors reported

that mare plasma Se concentrations were significantly greater in mares consuming

pasture and grain with no Se supplementation, and pasture and grain with Se

supplementation than in mares consuming pasture with no Se supplementation and

pasture with Se supplementation. Mares receiving Se supplement had significantly

greater plasma Se concentrations than mares receiving no supplement. Muscle and

colostrum Se concentrations were significantly greater in mares consuming pasture and

grain with Se supplementation and pasture with Se supplementation than in mares

32

consuming pasture with no Se supplementation, and pasture and grain with no Se

supplementation. No effect of treatment was reported on mare muscle or colostrum Se

concentrations. Mare plasma GSH-Px activities were not affected by nutrition or

selenomethionine supplementation. Foals of mares consuming pasture and grain with no

Se supplementation and pasture and grain with Se supplementation had significantly

greater plasma and muscle Se concentrations as compared to foals of mares consuming

pasture with no Se supplementation and pasture with Se supplementation. Foal plasma

GSH-Px activities were not affected by maternal plane of nutrition or selenomethionine

supplementation of the dam (Karren et al., 2010).

Brummer et al. (2011a) examined the effect of low Se status on the ability of both

the humoral and cell mediated components of the immune system to respond to a vaccine

challenge. Of the 28 horses used in the study, 7 received 0.14 ppm Se from sodium

selenite, and 21 horses received 0.07 ppm Se from sodium selenite. Blood samples were

taken at d 0 and thereafter every 4 wk for 28 wk, and analyzed for whole blood Se and

GSH-Px activity. Authors reported whole blood Se was significantly lower in horses

consuming 0.07 ppm Se (164.7 ng/mL) than horses consuming 0.14 ppm Se (211.1

ng/mL) after 28 wk of treatment. The authors also reported GSH-Px activity was

significantly lower in horses consuming 0.07 ppm Se (42.72 mU/mg Hb) than horses

consuming 0.14 ppm Se (55.00 mU/mg Hb). In response to vaccination, KLH-specific

Immunoglobulin G concentrations increased over time in both groups, but horses

consuming 0.14 ppm Se responded significantly quicker with significantly higher

antibody concentrations at 3 wk than horses consuming 0.07 ppm. Expression of the

33

transcription factor T-bet was significantly greater at 5 wk in horses consuming 0.14 ppm

Se horses consuming 0.07 ppm Se (Brummer et al., 2011a).

In a sister study, Brummer et al. (2011b) hypothesized that Se depletion would

result in a decrease in GSH-Px activity, serum total antioxidant capacity (TAC) and an

increase in oxidative stress, as measured as serum malondialdehyde concentration

(MDA), and T3:T4 ratio changes. The data indicated that whole blood Se concentration

significantly decreased over time in both groups but the decrease was greater in horses

consuming 0.07 ppm Se. At the end of 28 wk, T3:T4 significantly increased in horses

0.07 ppm Se, while it remained similar to initial levels in horses consuming 0.14 ppm Se.

Whole blood GSH-Px activity decreased during the study in both groups; however, final

whole blood GSH-Px activity was significantly different. Total antioxidant capacity did

not change during study. Malondialdehyde concentration was not different between

horses consuming 0.14 and 0.07 ppm Se at the initial or final draw, however MDA did

significantly increase over time in horses consuming 0.07 ppm Se while it remained

similar in horses consuming 0.14 ppm. The increased ratio of T3:T4 in 0.07 ppm Se

horses along with the changes in whole blood Se and GSH-Px suggested that horses

consuming 0.07 ppm Se were at, or approaching, deficient Se status (Brummer et al.,

2011b).

Podoll et al. (1992) fed 12 adult Arabian horses 1 of 2 treatments; 0.3 ppm

sodium selenite and 0.3 ppm sodium selenate to determine differences in supplemental Se

source. Serum was collected on d 0, 3, 7, 10, 14, 28, 42, and 49 for Se and GSH-Px

assays. The authors reported serum Se concentrations rose significantly during the trial in

34

all horses regardless of treatment. The overall response to supplemental Se was quadratic.

There were no significant differences observed in serum Se concentrations between those

consuming selenite or selenate. Serum GSH-Px activities were unaffected by the form of

Se but were significantly different over time (Podoll et al., 1992).

Montgomery et al. (2012a) assigned 15 Standardbred horses to 1 of 3 treatments;

a control receiving no supplementation, inorganic Se (sodium selenite), and organic Se

(Se yeast). Three months prior to trial, the study horses were turned out on a pasture

containing less than 0.05 ppm Se. The objective of the study was to examine the effects

of oral Se supplementation and Se source on aspects of innate and adaptive immunity in

horses. Immune function tests were performed that measured lymphocyte proliferation in

response to mitogen concanavalin A, and neutrophil phagocytosis, and antibody

production after rabies vaccination. Relative cytokine gene expression in stimulated

lymphocytes (interferon gamma, IL-2, IL-5, IL-10, tumor necrosis factor-alpha) and

neutrophils (IL-1, IL-6, IL-8, IL-12, tumor necrosis factor-alpha) was also examined.

Plasma and RBC Se, and blood GSH-Px activity were analyzed. Plasma and RBC Se

were significantly highest in horses in the organic Se group as compared to those in the

inorganic and control groups. Organic Se supplementation increased the relative

lymphocyte expression of IL-5, as compared to inorganic Se or no Se. Selenium

supplementation increased relative neutrophil expression of IL-1 and IL-8. Other

measures of immune function were unaffected (Montgomery et al., 2012a).

Montgomery et al. (2012b) also investigated the effect of dietary Se source on Se

status of mares and, consequently, the Se status and immune function of their foals. The

35

authors used 20 pregnant Standardbred mares. Mares were assigned to 1 of 2 treatments;

a complete pelleted feed containing 0.3 ppm organic Se, or a complete pelleted feed

containing 0.3 ppm inorganic Se. Mares were fed their respective diets for 2 mo prior to

estimated parturition. The authors reported that mean plasma Se concentrations prior to

the beginning of treatment were reflective of low Se intake, falling into a range

considered inadequate. Mare plasma and RBC Se concentrations increased in both groups

following onset of treatment. There was no significant effect of Se source on plasma or

RBC Se concentration. Se concentration in mammary secretion significantly declined

over time with the highest concentrations found in colostrum. No effect of Se source was

observed on colostrum or milk Se concentration measured at foaling, or during first mo

of lactation. In foals born to mares in the organic group, RBC Se concentration was 170%

compared to that of foals born to mares in inorganic group. However, source of maternal

Se did not influence IgG concentration in foals. At 1-d of age, foals in the organic group

had higher relative gene expression for interferon gamma. However, no significant

difference in relative gene expression of the neutrophil cytokines IL-1 and IL-8 at d 1 of

age was observed. Foals at 1-mo of age in the organic group had higher relative gene

expression for IL-2 when compared with foals in the inorganic group (Montgomery et al.,

2012b).

Pagan et al. (2007) used 4 mature trained Thoroughbred geldings in a 2-period

switch back design trial to evaluate how exercised Thoroughbreds digest and retain 2

forms of Se. Two horses were fed 2.90 mg inorganic Se (averaged 0.41 ppm Se with

about 77% selenite). The other two horses were fed 2.76 mg of organic Se yeast

36

(averaged 0.40 ppm Se with about 75% of total Se provided from yeast). In period 1,

respective diets were fed for 5 wk. The horses were exercised 6 d/wk in first 4 wk. In the

fifth wk, a 5-d digestion trial was conducted. On d 3 of collection, horses completed a

standardized exercise test. Feed, feces, urine and blood were analyzed for Se. In period 2,

Se supplementation was switched for 3 wk, horses received the same exercise in the first

2 wk, and in the third wk of total collections and an exercise test were conducted. The

authors reported horses consuming inorganic Se excreted significantly more fecal Se than

those consuming organic Se. Apparent absorption of dietary sodium selenite and organic

Se averaged 51.1and 57.3%, respectively. Selenium retention was increased when

organic Se was fed. The authors concluded most of the difference in Se retention was the

result of increased Se absorption, since there was no difference in average daily urinary

Se excretion between treatments. After the exercise test, horses consuming inorganic Se

had higher Se excretion as compared to d 1 or 2 of the collection period (Pagan et al.,

2007).

Richardson et al. (2006) fed 1 of 3 treatments to 18 sedentary 18-mo old stock

type horses; 11 geldings, and 7 mares. Treatments consisted of Control with no

supplemental Se, totaling 0.15 mg/kg Se, inorganic Se with control in addition to 0.45

mg/kg Se from sodium selenite, or organic Se with control in addition to 0.45 mg/kg Se

from Zn-L-selenomethionine to determine the effect of organic and inorganic Se sources

on the Se status of horses. Plasma and skeletal muscle Se concentrations and GSH-Px

activities in plasma, erythrocytes, and skeletal muscle were determined. Blood was drawn

on d 0, 28, and 56. Muscle samples were taken on d 0 and 56. The researchers reported

37

mean plasma and middle gluteal muscle Se concentrations on d 0 were not different

among treatments, and significantly increased over the experimental period. Plasma Se

concentrations were significantly greater on d 28 and 56 for Se-supplemented horses as

compared to control horses. There was a tendency for greater plasma Se concentrations in

horses consuming the organic treatment as compared to those consuming the inorganic

treatment on d 28. Mean muscle Se concentration was unaffected by treatment. Mean

plasma GSH-Px activity increased in horses consuming all treatments throughout the

trial. However, this activity was not affected by Se supplementation or source. Mean

erythrocyte GSH-Px activity also tended to increase over the experimental period for

horses fed all diets. There was a tendency for horses consuming the organic treatment to

have greater erythrocyte GSH-Px activity on d 28 as compared with those consuming

both the control and inorganic treatments. The authors hypothesized the rapid (less than 4

wk) increase in the erythrocyte GSH-Px activity of horses consuming the organic diet

may indicate greater incorporation into erythrocyte GSH-Px. Mean erythrocyte GSH-Px

activity of horses consuming inorganic and organic treatments were not different as

compared to those consuming control on d 56. Mean skeletal GSH-Px activity

significantly decreased over the experimental period for all horses (Richardson et al.,

2006).

Richardson et al. (2003) sought to determine the effects of Se source and Se status

in horses. These researchers compared Se concentrations and GSH-Px activities in blood

and skeletal muscle of horses receiving organic and inorganic Se supplementation.

Twenty-four 16-mo-old horses were fed 1 of 4 treatments: control containing 0.15 mg

38

Se/kg, inorganic containing 0.6 mg/kg sodium selenite, organic treatment 1 containing

0.6 mg Se/kg, organic treatment 2 containing 0.6 mg Se/kg. All horses received the basal

diet during a 28-d acclimation period, and were placed on their respective treatments for

a 56-d supplementation period. Blood was drawn on d 0, 28, and 56 of the

supplementation period. Plasma was harvested and the RBC fraction was washed and

lysed. Muscle biopsies were taken from the middle gluteal muscle on d 0 and 56. The

authors reported plasma Se concentrations of the supplemented groups significantly

increased from d 0 to 28, plateaued by d 56, and were significantly greater than control

on d 28 and 56. Mean plasma Se concentration of those consuming organic treatment 1

was greater than those consuming organic treatment 2 and inorganic on d 28, and

continued to be greater than those consuming organic treatment 2 on d 56, and tended to

be different from inorganic on d 56. Muscle Se concentrations increased in horses on all

treatments from d 0 to 56. Plasma GSH-Px activity fluctuated over time, but was not

affected by treatment. Erythrocyte GSH-Px activity significantly increased between d 0

and 28 in organic treatment 1 and was significantly greater than the other 3 treatments on

d 28 (Richardson et al., 2003).

Janicki et al. (2001) used 15 mares to determine if Se form or level had an effect

on mare and foal Se status, GSH-Px activity, and antibody titer to influenza. The mares

were blocked by expected foaling date and assigned to 1 of 3 treatments; 1 mg sodium

selenite, 3 mg sodium selenite, or 3 mg Se-yeast. The respective diets were fed for 55 d

pre-foaling, to 56 d post-foaling. Mare blood samples were taken prior to

supplementation, every 2 wk until foaling, immediately post-foaling, and every wk for 56

39

d. Colostrum samples were taken post-foaling, and milk samples every 2 wk for 56 d.

Foal blood samples were obtained prior to suckling, at 12 h, and 2, 4, 6, and 8 wk of age.

Selenium concentration, GSH-Px activity and serum influenza antibody titers were

analyzed. Serum Se was significantly greater in mares receiving 3 mg organic Se as

compared to other treatments at post-foaling, wk 4 and 8. Selenium in colostrum and milk

was greater in mares receiving 3 mg organic Se as compared to other treatments. At 12 h,

serum Se in foals from mares receiving organic Se was significantly greater than foals

from mares receiving 1 mg inorganic Se. At 2, 4, 6, and 8 wk, serum Se in foals from

mares receiving organic was significantly greater than those receiving other treatments.

At 2 wk, serum Se was also significantly greater in foals from mares receiving 0.3 ppm

inorganic as compared to foals from mares receiving 0.1 ppm inorganic. At 6 wk, GSH-

Px activity in foals from mares receiving 0.3 ppm was significantly greater than foals

from mares receiving 0.1 ppm. At 8 wk, GSH-Px activity was significantly greater in

foals from mares receiving 0.3 ppm organic Se compared to foals from mares receiving

0.1 ppm inorganic Se. At 6 wk, influenza antibodies to A2/KY/92 were significantly

greater in foals from mares receiving 0.3 ppm than foals from mares receiving 0.1 ppm.

At 2, 4, and 8 wk, influenza antibodies to A2/KY/92 tended to be greater in foals from

mares receiving 0.3 ppm. At 8 wk, influenza antibodies to A1/Prague were significantly

greater in foals from mares receiving 0.3 ppm than in foals from mares receiving 0.1 ppm

(Janicki et al. 2001).

White et al. (2011) hypothesized that Se supplementation above NRC

recommendations would enhance selenoprotein activity and reduce oxidative damage in

40

horses following a prolonged exercise bout. Twelve mature, untrained Thoroughbreds

were fed 1 of 2 respective diets for 36 d; 0.1 mg/kg sodium selenite or 0.3 mg/kg sodium

selenite. On d 35, horses were subjected to 120 min of submaximal exercise with a mean

heart rate of 135 bpm. Blood samples were taken at d 0, after 34 d of Se supplementation,

and on d 35 immediately after exercise; and 6 and 24 h post-exercise. Samples were

analyzed for serum Se, plasma and RBC lysate GSH-Px activity, serum creatine kinase,

and total lipid hydroperoxides. Muscle biopsies were taken d 0 and after 34 d of Se

supplementation; and at 6 and 24 hr post-exercise on d 35 and 36 for determination of

GSH-Px and thioredoxin reductase activities. The authors reported supplementation with

0.3 ppm significantly increased serum Se, but had no effect on GSH-Px activity in

plasma, RBC lysate or muscle in horses at rest. Serum creatine kinase was not different

between horses consuming 0.3 ppm and 0.1 ppm, but significantly increased in response

to prolonged exercise, indicating excessive reactive oxygen species generation and tissue

damage. Serum lipid hydroperoxidase was significantly affected in horses fed 0.3 ppm,

indicating these horses were possibly better equipped to combat the oxidative load.

Glutathione peroxidase activity significantly increased in plasma and significantly

decreased in RBC lysate after prolonged exercise in all treatments. A significant

treatment by time interaction was observed for RBC lysate and muscle GSH-Px activity.

Compared to enzyme activity before exercise, RBC GSH-Px activity was significantly

lower immediately after exercise in horses fed 0.3 ppm, whereas a similar decline wasn’t

significantly observed until 6 h post-exercise in those consuming other treatments.

Muscle GSH-Px activity was significantly elevated over pre-exercise levels at 6 h post-

41

exercise in those consuming 0.3 ppm and remained unchanged in horses consuming 0.1

ppm (White et al., 2011).

Shellow et al. (1985) fed 20 mature geldings; 10 Quarter Horses and 10

Thoroughbreds to determine the influence of dietary Se on whole blood and plasma Se

levels, and GSH-Px activity. All horses were fed a basal diet of 50% concentrate

containing 0.077 mg/kg of naturally occurring Se, 50% Timothy hay containing 0.43

mg/kg of naturally occurring Se for a total of 0.060 mg/kg of Se for at least a 4-wk

preliminary period. At the beginning of the repletion phase, horses were supplemented

with 0, 0.05, 0.1, or 0.2 ppm Se as sodium selenite with final Se concentrations of 0.06,

0.11, 0.16, and 0.26 ppm. Blood was drawn at weekly intervals for 2 wk before

supplementation, and at 12 wk following inclusion of supplemental Se in diet. The

authors reported a significant increasing linear trend in plasma Se concentration over

time. At wk 0, there was no significant difference observed among treatment groups.

Supplementation of the diets with Se significantly increased plasma Se above that of the

control group by the wk 2 of the trial. By wk 5, there were significant differences in

plasma Se concentration between horses in the control group, and those receiving 0.05,

0.10, or 0.2 ppm supplemental Se in their diet. Plasma Se concentrations for horses

receiving the 2 highest levels of Se were significantly greater than those receiving 0.05

ppm supplemental Se. There were no significant differences in plasma Se concentrations

between those receiving 0.10 and 0.20 supplemental Se. Little change in plasma Se

concentration was observed in Se-supplemented horses after 5 wk. Plasma Se reached

plateaus of 0.1 to 0.11, 0.12 to 0.14, and 0.13 to 14 µg/mL in horses supplemented with

42

0.5, 0.1, and 0.2 ppm Se, respectively. Maximum response in whole blood Se

concentration occurred by wk 6 with no further significant changes throughout the

remainder of the trial. Whole blood Se reached plateaus of 0.16 to 0.18, 0.19 to 0.21, and

0.17 to 0.18 µg/mL in groups supplemented with 0.05, 0.1, and 0.2 ppm Se, respectively.

Plasma GSH-Px activity was not significantly affected by dietary treatment, although an

increasing trend in activity over time was observed (Shellow et al., 1985).

Calamari et al. (2010) compared the effects of organic and inorganic Se

supplements on hematological profiles, enzyme activities, plasma oxidative status, and

inflammatory status. Twenty-five slightly exercised, mature Italian Saddle Horses were

used in the trial. All horses were fed the control diet for 56 d to allow for diet adaption.

The trial utilized 5 treatments; negative control, 0.2 mg organic Se/kg, 0.3 mg organic

Se/kg, 0.4 mg organic Se/kg, or positive control containing 0.3 mg inorganic Se/kg.

Blood was drawn d 0, 28, 56, 84, and 112. Authors reported plasma metabolites related to

energy and protein metabolism and mineral metabolism were not affected by Se source or

dose. Inflammatory status did not appear to be affected by Se source and dose. Horses

consuming 0.3 mg organic Se and 0.4 mg organic Se had significantly lower total plasma

antioxidants than horses consuming control, and horses consuming Se yeast supplement

had significantly lower total plasma antioxidants as compared to those consuming

comparable dose of selenite. Total plasma antioxidants decreased linearly as Se yeast

supplementation increased. Total white blood cells was not affected by treatment.

Number of lymphocytes tended to increase slightly as Se-yeast supplementation

increased. Greater numbers of lymphocytes were observed in those consuming 0.3 mg

43

organic Se and 0.4 organic Se as compared to those consuming 0.3 mg inorganic Se

(Calamari et al., 2010).

In a companion study to Calamari et al. (2010), Calamari et al. (2009) evaluated

the effects of dietary Se sources on Se status, GSH-Px activity, and thyroid hormone

status. Blood was analyzed for RBC GSH-PX activity, whole blood Se, packed cell

volume, and plasma Se concentrations. The authors reported horses consuming all

treatments supplemented with Se had significantly greater total Se concentrations in

whole blood and plasma when compared with those consuming the negative control. A

linear dose effect and source effect were observed for total Se in blood. Whole blood Se

concentrations were significantly higher in treatments supplemented with greater doses of

Se, and in those horses supplemented with Se yeast at d 84 and 112 as compared to those

receiving a comparable dose of selenite. Total Se in blood in horses consuming all

treatments supplemented with Se was greater as compared to those consuming the control

from d 28 to the end of the study. The 16-wk experimental trial was not sufficient for

horses consuming all treatments to achieve asymptotic, steady-state Se concentrations in

whole blood. There was a significant linear dose effect for plasma Se, with greater values

in those consuming treatments supplemented with greater doses of Se. Selenium source

did not affect plasma Se concentrations. Plasma Se concentrations achieved asymptotic

steady state within 75 to 90 d of the beginning of the study in all supplemented groups.

Plasma Se in all treatments appeared to increase by 50 to 60% at d 28, 85 to 93% at d 56,

and almost 100% at d 84. Correlations were observed between whole blood Se and

plasma Se (r = 0.83). Horses consuming all treatments supplemented with Se had

44

significantly greater GSH-Px activity when compared with those consuming the negative

control. Linear and quadratic dose effects were observed on GSH-Px activity between

those consuming low and intermediate doses of Se yeast, or between those consuming the

least and greatest dose of Se yeast. Horses consuming all supplemented treatments had

significantly greater GSH-Px activity as compared to those consuming negative control

from d 56 to study completion. Asymptotic GSH-Px activity did not appear to have been

achieved in any of the horses consuming Se-supplemented treatments after completion of

the 16-wk experimental period. There was a correlation observed between GSH-Px

activity and whole blood Se (r = 0.86), and GSH-Px activity and plasma Se

(r = 0.75). Plasma GSH-Px activity was significantly greater in those consuming Se yeast

and sodium selenite when compared with those consuming the negative control. The rate

of increase in the proportion of total Se as selenomethionine over time was significantly

greater in whole blood and plasma in those horses consuming 0.3 mg organic Se as

compared with those consuming a comparable dose of selenite. Selenocysteine was the

predominant form of Se in blood and accounted for 79.1 and 71.4% of total Se in whole

blood and plasma, respectively, whereas selenomethionine only accounted for 15.2 and

10.0% (Calamari et al., 2009).

Brummer et al. (2013) evaluated the impact of change in Se status on measures of

antioxidant status and oxidative stress in adult horses during Se depletion and repletion.

Twenty-eight horses were divided into 4 treatment groups. During the 196-d depletion

period, 3 treatments provided 0.06 mg Se/kg DM and 1 treatment provided 0.12 mg

Se/.kg DM. During the 189 d repletion period, horses were assigned to 1 of 4 treatments;

45

7 horses continued to consume 0.06 mg Se/kg, 7 horses continued to consume 0.12 mg

Se/kg, 7 horses were fed Se-yeast at 0.3mg/kg, and 7 horses were fed sodium selenite

0.3mg/kg. Horses were not exercised during this trial. The 196-d depletion period was

selected on the basis of the Se status of the horses as determined by the monthly blood

samples obtained for whole blood Se and GSH-Px activity evaluation and compared with

published adequate reference range for whole blood Se between 180 to 240 ng/mL, and

whole blood GSH-Px activity between 40 to 160 enzyme units/g Hb (Stowe, 1998).

Blood samples were taken at the start of each phase and on d 84, 140, 168, and 196 of

depletion and d 28, 56, 154, and 189 of repletion. The authors reported whole blood Se

concentrations were affected by the interaction of treatment and time. The authors also

appeared to erroneously report significant main effects of treatment and time during the

depletion phase. Whole blood Se concentrations in horses consuming 0.06 mg Se

decreased until d 140 then stabilized and were significantly less than those consuming

0.12 mg Se. Selenium concentrations in horses consuming 0.12 mg Se stabilized within

first 84 d of depletion. At the end of depletion, there was a significant difference in whole

blood Se between those consuming the 2 treatments. Whole blood GSH-Px activity in

those consuming 0.12 mg Se decreased during first 84 d and then stabilized. Glutathione

peroxidase activity in those consuming 0.06 mg Se decreased throughout the depletion

period. Mean GSH-Px activity was less in those consuming 0.06 mg Se as compared to

those consuming 0.12 mg Se at d 196. A positive correlation existed between whole

blood Se and GSH-Px activity (r = 0.63). During repletion, there was a significant

treatment by time interaction. The authors also appeared to erroneously report significant

46

main effects of treatment and time. Within 28 d of starting the repletion phase, whole

blood Se was similar in those consuming 0.12 mg/kg Se, 0.3 mg/kg organic Se, and 0.3

mg/kg inorganic Se, but greater than those consuming 0.06 mg Se/kg DM. At 154 d,

whole blood Se concentrations in those consuming 0.3 mg organic Se/kg DM and 0.3 mg

inorganic Se/kg DM were significantly greater than those consuming 0.12 mg Se/kg DM.

Whole blood Se did not increase from d 154 to 189 in either those consuming 0.3 mg

inorganic Se or 0.3 mg organic Se. Whole blood GSH-Px activity during the repletion

phase was affected by the interaction of treatment by time. The authors also appeared to

erroneously report main effects of treatment and time. At d 154, GSH-Px activity in those

consuming 0.3 mg inorganic Se were comparable to those consuming 0.3 mg organic Se,

and appeared greater than those consuming 0.12 mg Se. A strong positive correlation

existed between whole blood Se and GSH-Px activity (r = 0.82). The authors theorized

the current Se recommendation of 0.1 mg Se/kg DM must be close to the minimum Se

requirement for mature idle horses. The authors stated that whole blood GSH-Px is

responsive to dietary Se intakes above 0.1 mg/kg and supplementation of 0.1 ppm Se may

not allow for maximum GSH-Px activity in the horse. An increase in whole blood GSH-

Px activity required 56 d of repletion in comparison with a response time of 28 d of

repletion for whole blood Se. These differences in response times are most likely due to

the incorporation of GSH-Px in recently formed red blood cells. In this study, both the

depletion and repletion phases exceeded the period needed for the complete turnover of

the RBC population, thus allowing enough time for the incorporation of GSH-Px into

recently formed RBC. The authors suggested that the lack of detectable change in GSH-

47

Px activity in response to supplementation levels above 0.1 mg/kg DM could be due to

the shorter experimental periods used in some studies compared with the length of time

required for complete RBC turnover in the horse (Brummer et al., 2013).

Statement of the Problem

Selenium deficiency in horses is a prominent problem in the Pacific Northwest,

and other areas with extremely deficient soils. Although the current NRC

recommendation is 0.1 ppm (NRC, 2007), studies have reported conflicting results on the

possible benefits of 0.3 ppm Se supplementation. Data from previous studies indicate that

in order to see a benefit of 0.3 Se supplementation, trials need to be conducted for at least

112 d, and whole blood Se concentrations and erythrocyte GSH-Px activity should be

used to evaluate Se status.

Little data exists as to the rate of Se depletion in horses consuming Se-deficient

diets. Brummer et al. (2013) drew blood on d 0, 84, 140, 168, and 196 d of a depletion

period. It is difficult to determine a precise depletion curve with so much time in between

blood sampling. The objectives of the current study was to 1) determine the depletion

rate of Se in horses consuming a Se-deficient diet and 2) compare the effects of two

different levels of organic Se supplementation on Se repletion as indicated by whole

blood Se concentrations and erythrocyte GSH-Px activity in moderately exercised horses.

48

Chapter III

MATERIALS AND METHODS

Experimental Design

Twelve mature, stock-type geldings were used in a 2-part study. First, to

determine the effects of feeding a low-Se diet, containing 23% of the NRC recommended

amount of dietary Se, on whole blood Se concentrations and erythrocyte glutathione

peroxidase (RBC GSH-Px) activity over a 112-d depletion phase. Secondly, the geldings

previously depleted to an average whole blood Se concentration of 109 ng Se/mL

received 1 of 2 levels of Se organic supplementation, in an effort to compare the rate of

repletion between horses consuming a supplement containing 0.1 vs. 0.3 ppm organic Se.

Horses were divided into 4 groups of 3 and housed in 6 x 20 m pens at the West

Texas A&M University Horse Center. Throughout the trial, horses were classified as

moderately exercised (NRC, 2007), as they were used in horsemanship classes and

equestrian team practices 3 to 5 times/ wk. Horses were fed individually in 2 x 5 m stalls

twice daily at 0600 and 1700, and were allowed 3 h to consume rations before being

turned out in 6 x 20 m pens. Supplement and hay was weighed out prior to feeding.

Intakes and orts were weighed and recorded throughout the trial. Routine farrier work,

vaccinations, and deworming were consistent with West Texas A & M University

protocols. Salt blocks were provided ad libitum throughout the study. Body condition

49

scores were assigned, and BW was measured at 0500 in 28-d intervals on a platform scale

(LBS Inc. Garden City, KS). Jugular venous blood was drawn on d 0, 28, 56, 84 and 112

of the depletion phase, and on d 14, 28, 56, 84, 96 and 112 of the repletion phase at 0500.

On day 2 of the depletion phase, one gelding died due to natural causes unrelated to the

study. On d 8 of the depletion phase, another gelding was removed from the trial due to

refusal to consume the supplement. During the repletion phase, 10 horses were stratified

by whole blood Se concentrations at d 84 of depletion, and evenly assigned to 1 of 2

repletion treatments. The 10 remaining horses ranged in age from 9 to 19 yr with a mean

age of 14 yr. Trial protocol was approved by West Texas A & M University Institutional

Animal Care and Use Committee.

Diets

At the onset of the trial (d 0), venous blood was drawn, weights recorded, and

BCS assigned. Horses were fed the depletion diet for 112 d. During the repletion phase,

horses were fed their respective treatments for 112 d. Diets were fed in amounts to

attempt maintenance of BCS of 5.0.

All horses consumed a basal diet of Orchard Grass Hay fed at 1.25 to 2.34%

BW/d. Hay was grown in extremely Se-deficient soils (< 0.5 ppm soil Se; Koller and

Exon, 1986) in Central Oregon. In addition the hay was fertilized with (NH4)2SO4, a

commonly used fertilizer, which decreases Se uptake into the plant due to the

antagonistic relationship between S and Se. During the depletion phase, diets consisted

of the Orchard Grass Hay top dressed with 57 g of vitamin/mineral supplement with no

added Se. At d 0 of the repletion phase, horses were stratified by whole blood Se

50

concentrations at d 84 of depletion and then evenly divided and assigned one of two

supplemental Se treatments. Diets consisted of the Orchard Grass Hay along with

supplemental Se contained in the vitamin/mineral supplement that was top-dressed at 2

concentrations; 0.1 ppm Se (SE1; n = 5); or 0.3 ppm Se (SE3; n = 5).

Hay was analyzed for DE, CP, ADF, and NDF. Feed analysis for Orchard Grass

Hay is presented in Table 1. Samples of all supplements (No Se, SE1 and SE3) and hay

were analyzed for Se concentration at the Michigan State University Diagnostic Center

for Population and Animal Health (DCPAH; Lansing, MI; Table 2)).

Table 1. Feed Analysis for Orchard Grass Hay (DM)

Crude Protein, % 13.1

Acid Detergent Fiber, % 39.1

Neutral Detergent Fiber, % 58.2

DE Mcal/kg 2.18

Table 2. Selenium Analysis for Supplements and Hay (DM)

Orchard Grass Hay (mg/kg) 0.01

No Se Added Supplement (mg/1.9 oz) 0.14

1 ppm Se Supplement (mg/1.9 oz) 1.05

3 ppm Se Supplement (mg/1.9 oz) 3.52

Sample Collections, Preparation, and Handling

Venous blood samples were collected at 0500 prior to the morning feeding on d 0,

28, 56, 84, 112 of the depletion phase, and d 14, 28, 56, 84, 96 and 112 of the repletion

phase via jugular veni-puncture using two 3-ml lavender-top Vacutainer™ tubes

containing EDTA. After blood collection, sample tubes were slowly inverted 8 times, and

then placed on ice. One tube of whole blood from each horse was shipped on ice at 0900

51

in specialized insulated containers purchased from DCPAH (Lansing, MI) to DCPAH

(Lansing, MI) for Se analysis. The remaining tubes were transported to the West Texas A

& M University CORE laboratory (Canyon, TX) to be prepared for GSH-Px analysis and

stored. For GSH-Px analytical preparation, four 500 uL whole blood samples from each

tube were transferred into 2 mL micro-centrifuge tubes. The 2 mL tubes were centrifuged

at 2500 x g for 5 min. After separation, plasma was discarded. Remaining RBC were

washed with 500 uL of 0.9% NaCl solution, vortexed, and centrifuged again at 2500 x g

for 5 min. Saline supernatant was removed and discarded. Erythrocytes were lysed with 1

mL of ice-cold distilled, deionized water, vortexed, closed and stored upright at -80◦C

until RBC GSH-Px activity analysis.

Laboratory Analysis

Inductively-Coupled Plasma Mass Spectrometry. An inductively-coupled plasma

mass spectrometry (ICP-MS) 7500ce (Agilent Technologies, Santa Clara, CA) was used

to determine concentrations of Se in whole blood and feed samples at DCPAH (Lansing,

MI). For preparation of whole blood Se analysis, 200 uL of whole blood was mixed with

5 mL of diluent containing; NH4OH, butanol, EDTA, Triton-x 100, and 5 internal

standards. Samples were analyzed for Se using the ICP-MS on “non-gas” mode, and Se

concentration were reported in ng Se/mL whole blood.

Glutathione Peroxidase Activity Assay. For the analysis of GSH-Px activity, RBC

samples were thawed in the West Texas A&M University CORE Laboratory (Canyon,

TX) and analyses performed in the West Texas A&M University RHIL Laboratory

(Canyon, TX) using an EPIC spectrophotometer (Palmyra, WI). The spectrophotometer

52

was set to the “kinetic option”, and GSH-Px activity was determined at a wavelength of

340 nm. The reading were collected every 30 s for 3 min. Each well of the assay kit

contained 75 uL assay buffer, 75 uL NADPH reagent, and 15 uL diluted sample.

Erythrocyte samples were diluted using 7 uL sample and 64 uL assay buffer, and were

then plated in their respective wells, running all blood draw from each respective horse

on the same plate twice. Two controls were created, a high (225 mU/ mL), and low

control (112.5 mU/ mL). Blank standard, low control and high control were then plated

using 15 uL of each. Using the multi-channel pipette, 75 uL tert-butyl was added to each

column (12 columns per plate), and the respective column analyzed.

Once samples were analyzed, the rate of decrease in absorbance at 340 nm per

min was calculated. The net rate for the sample was calculated by subtracting the rate

observed for the water blank. The net absorbance/min was calculated as:

1 mU/mg Hb = 1 nmol NADPH/mL = (A340/min)/ 0.00622

The concentrations were then corrected for the dilution of the sample (10:90

dilution), and the dilution of the RBC and deionized water (1:5). The units of activity in

original sample are expressed mU/mg Hb.

Statistical Analysis

Data for depletion phase whole blood Se concentrations and RBC GSH-Px

activity was analyzed using non-linear regression analysis (SPSS Version 21, 2012). Data

for repletion phase whole blood Se concentrations were adjusted by subtracting d 112 of

53

depletion values from all other days of repletion for each horse to determine changes in

whole blood Se concentrations, and was analyzed using non-linear regression analysis

(SPSS Version 21, 2012). The slope of the non-linear regression curves were compared

using the t-test. Erythrocyte GSH-Px activity was also analyzed using non-linear

regression analysis (SPSS Version 21, 2012). Data for whole blood Se concentrations

was also analyzed using the t-test assuming equal variances (Excel, 2010) to determine

differences between treatments within time. Data for repletion phase RBC GSH-Px

activity was analyzed using t-tests assuming unequal variances (Excel, 2010). Significant

differences between treatments were declared at P ≤ 0.05. Trends for differences between

treatments were declared at P ≤ 0.10.

54

Chapter IV

RESULTS AND DISCUSSION

Mean Se intakes for horses consuming Se depletion diet, 0.1 ppm supplemental

Se treatment (SE1), and 0.3 ppm supplemental Se treatment (SE3) are presented in Table

3. During the depletion phase, horses consuming overall mean of 23% of NRC

recommendations for Se. Horses consuming SE1 consumed an overall mean of 131% of

NRC recommendations for Se. Horses consuming SE3 consumed an overall mean 421%

of NRC recommendations for Se.

Table 3. Mean Selenium Intake (mg/kg DM)

Se depletion 0.023

SE1 0.131

SE3 0.421

Depletion Phase Whole Blood Selenium Concentrations Regression

At initiation of the depletion phase (d 0), overall mean whole blood Se

concentrations were 187.4 ± 7.36 ng Se/mL. Individual whole blood Se concentrations

during the depletion phase can be observed in Figure A-1 in the Appendix. Overall whole

blood Se concentrations in horses consuming 23% of NRC Se recommendations depleted

at a non-linear rate. A non-linear regression equation was developed {predicted whole

55

blood Se concentration = 184.95 * (1 * EXP (-0.005 * day))} and can be observed in

Figure 1. The corrected r-squared of Se depletion was 0.863.

Using the non-linear equation, forecasting of whole blood Se concentrations was

estimated to d 250 (Figure 2). If the predicted equation was proven, horses consuming

23% of NRC recommendations for Se would become clinically deficient within 209 d

from initiation of the depletion period. Non-linear regression equations were developed

because of biological reasons in the body. The rate of depletion slows over time. Linear

regression equations would possibly predict clinical Se deficiency too quickly.

There are no published studies reporting the non-linear regression of a Se depletion

phase. However, data for Se depletion in horses has been reported. Brummer et al. (2013)

reported horses fed 60% of the NRC recommendation of Se for 196 d had significantly

lower whole blood Se concentrations at d 140 and 196 as compared to horses fed 0.12 mg

Se/kg DM. Furthermore, Brummer et al. (2013) reported significantly lower whole blood

Se concentrations in horses fed 0.06 mg Se/kg DM at d 84, 140, 168, and 196 as

compared to d 0. Whole blood Se concentrations in horses receiving 0.06 mg Se/kg DM

was significantly lower at d 140, 168, and 196 as compared to d 84. However, the authors

reported no significant differences in whole blood Se concentrations between d 140, 168

and 196. In the current study, overall mean whole blood Se concentrations at the

initiation of depletion were 187.4 ng Se/mL, as compared to Brummer et al. (2013), who

reported overall mean whole blood Se concentrations of 251.7 ng Se/mL. In addition, at

the end of the depletion phase (d 112) in the current study, overall mean whole blood Se

concentrations in horses consuming 23% of NRC recommendations were 109 ng Se/mL.

58

Brummer et al. (2013) reported whole blood Se concentrations in horses consuming 60%

of NRC recommendations of 173.5 ng Se/mL at d 140, and 165.1 ng Se/mL at d 196.

Repletion Phase Whole Blood Selenium Concentrations Regression

At the initiation of the repletion phase (d 112 of depletion), horses were stratified

according to whole blood Se concentrations at d 84 of depletion, and assigned to 1 of 2

Se supplement treatments. Horses assigned to SE1 had overall mean whole blood Se

concentrations of 108.2 ± 12.2 ng Se/mL. Horses assigned to SE3 had overall mean

whole blood Se concentrations of 109.8 ± 11.2 ng Se/mL. Individual whole blood Se

concentrations during the repletion phase can be observed in Figure A-2 in the Appendix.

Non-linear regression equations were developed using adjusted whole blood Se

concentrations, calculated by subtracting d 0 of repletion values from d 14, 28, 56, 84, 96

and 112. Adjusted whole blood Se concentrations in horses consuming SE1 repleted at a

non-linear rate. A non-linear regression equation was developed {predicted change in

whole blood Se concentration = 20.911 * (1 - EXP (-0.062 * day))} and is shown

graphically in Figure 3. The corrected r-squared of Se repletion in horses consuming SE1

was 0.550.

Adjusted whole blood Se concentrations in horses consuming SE3 also repleted at

a non-linear rate. A non-linear regression equation was developed {predicted change in

whole blood Se concentration = 38.249 * (1 - EXP (-0.070 * day))} and is shown

graphically in Figure 4. The corrected r-squared of Se repletion in horses consuming SE3

was 0.779.

61

When comparing the non-linear regression equations of the change in whole

blood Se concentrations in horses consuming SE1 and SE3, it appeared that horses

consuming SE3 repleted at a faster rate, and maintained higher whole blood Se

concentrations. Non-linear regressions over 112-d Se repletion are shown in Figure 5.

There are no studies reporting non-linear regression analysis of whole blood Se

concentrations during a repletion phase. However, Calamari et al. (2009) reported linear

regressions of whole blood Se concentrations {total blood Se, ng/g = 1.472 ± 0.278 x

time (d) + 179.8 ± 19.1} in horses consuming 0.18 mg Se yeast/kg DM, {total blood Se,

ng/g = 2.186 ± 0.267 x time (d) + 195 ± 19.7} in horses consuming 0.29 mg Se yeast/kg

DM, { total blood Se, ng/g = 2.167 ± 0.301 x time (d) + 232.6 ± 23.3} in horses

consuming 0.39 mg Se yeast/kg DM, and {total blood Se, ng/g = 2.186 ± 0.267 x time (d)

+ 195 ± 19.7} in horses consuming 0.29 mg Na selenite/kg DM. Calamari et al. (2009)

also reported a quadratic regression for plasma Se concentrations {plasma Se, ng/g = -

0.00697 ± 0.00537 x time (d)2 + 1.2991 ± 0.6269 x time (d) + 97.1 ± 14.8} in horses

consuming 0.18 mg Se yeast/kg DM, { plasma Se, ng/g = -0.01727 ± 0.00356 x time (d)2

+ 2.5768 ± 0.4157 x time (d) + 89.2 ± 9.8} in horses consuming 0.29 mg Se yeast/kg

DM, { plasma Se, ng/g = -0.01556 ± 0.00417 x time (d)2 + 2.4917 ± 0.4865 x time (d) +

104.2 ± 11.5} in horses consuming 0.39 mg Se yeast/kg DM, and { plasma Se, ng/g = -

0.01478 ± 0.00443 x time (d)2 + 2.2985 ± 0.5178 x time (d) + 81.2 ± 12.2} in horses

consuming 0.29 mg Na selenite/kg DM.

63

In the current study, non-linear regression equations were developed because of

biological reasons in the body. The rate of repletion slows over time. Linear regression

equations would possibly predict clinical Se deficiency too quickly.

Upon analysis of the repletion data, a decrease in whole blood Se concentrations

appears to occur at d 84 in both SE1 and SE3, before returning to expected values at d 96

and 112. Overall mean whole blood Se concentrations at d 84 were below concentrations

at d 28 and 56. The reason for this dramatic, and unexpected, decrease in Se

concentrations at d 84 of repletion is unknown. Possible causes include a difference in

sample handling during shipment of samples to DCPAH, or differences, however slight,

in laboratory analysis of the samples.

However, d 84 data from this study partially agree with Shellow et al. (1985) who

reported horses consuming 0.16 ppm Se had whole blood Se concentrations of 0.140 ug

Se/mL at d 56, and had decreased concentrations of 0.138 ug Se/mL at d 63, although this

decrease was not statistically significant. Further, the authors reported horses consuming

0.26 ppm Se had whole blood Se concentrations of 0.142 ug Se/mL at d 63, and these

values declined slightly to 0.135 ug Se/mL at d 70, although again, the decrease was not

statistically significant.

Whole Blood Selenium Concentrations in Horses Consuming 0.1 and 0.3 ppm Selenium

at d 0 of Repletion

There was no significant effect of treatment observed on overall mean whole

blood Se concentrations in horses consuming SE1 (mean = 108.2 ng Se/mL) and SE3

(mean = 109.8; at d 0 of repletion (P = 0.417; Figure 6).

65

Data for whole blood Se concentrations at d 0 of repletion in the current study

agree with that of Brummer et al. (2013), who reported no differences between horses

previously fed 0.06 mg Se/kg DM for 196 d at d 0 of repletion. The results also agree

with Calamari et al. (2009), who reported no differences in whole blood Se concentration

in horses fed 0.085 mg Se/kg DM for 2 mo at d 0 of repletion. Richardson et al. (2003)

and Richardson et al. (2006) reported no significant differences at d 0 of repletion in

plasma Se concentrations in horses fed 0.15 mg Se/kg DM for 28 d. The d 0 of repletion

results of this study disagree with Shellow et al. (1985), who reported significantly lower

whole blood Se concentrations in horses fed 0.06 ppm Se for at least 4 wk, in horses

consuming 0.06 and 0.26 ppm Se as compared to horses consuming 0.16 ppm Se, and

significantly higher whole blood Se concentrations in horses consuming 0.11 ppm Se as

compared to horses consuming 0.26 ppm Se.


at d 14 of Repletion

A significant effect of treatment was observed on overall whole blood Se

concentrations in horses consuming SE1 and SE3 at d 14 (Figure 7). Horses consuming

SE3 (mean = 129.8 ng Se/mL) had significantly greater (P = 0.032) whole blood Se

concentrations as compared to horses consuming SE1 (x bar = 115.6 ng Se/mL).

Data for whole blood Se concentrations at d 14 of the current study partially agree

with that of Shellow et al. (1985), who reported significantly lower whole blood Se

concentrations at d 14 in horses consuming 0.06 and 0.11 ppm Se as compared to horses

consuming 0.16 ppm Se. However, these authors also reported significantly higher whole

67

blood Se concentrations in horses consuming 0.16 ppm Se as compared to horses

consuming 0.26 ppm Se. The results of the current study disagree with Janicki et al.

(2001), who reported no difference in serum Se concentrations at d 14 of supplementation

in pregnant mares supplemented with 1 or 3 mg Se/d.






concentrations as compared to horses consuming SE1 (mean = 129.4 ng Se/mL).

Data for whole blood Se concentrations at d 28 of the current study agree with

Richardson et al. (2006), who reported significantly higher plasma Se concentrations in

horses consuming 0.45 mg organic and inorganic Se/kg DM as compared to horses

consuming 0.15 mg Se/kg DM. Additionally, these results agree with Richardson et al.

(2003), who reported plasma Se concentrations were significantly greater at d 28 in

horses consuming 0.6 mg organic Se/kg DM as compared to horses consuming 0.15 mg

Se/kg DM. The results of this study partially agree with that of Shellow et al. (1985), who

reported significantly higher whole blood Se concentrations at d 28 in horses consuming

0.11 ppm Se, 0.16 ppm Se, and 0.26 ppm Se as compared to horses consuming 0.06 ppm

Se. Data from this study also partially agrees with Calamari et al. (2009) who reported

horses consuming 0.18 mg organic Se/kg DM, 0.29 mg organic Se/kg DM, 0.39 mg

organic Se/kg DM, and 0.29 mg inorganic Se/kg DM had significantly greater whole

69

blood Se concentrations as compared to horses consuming 0.085 mg Se/kg DM.

Furthermore, the researchers reported horses consuming 0.39 mg organic Se/kg DM, and

0.29 mg inorganic Se/kg DM had significantly greater whole blood Se concentrations as

compared to horses 0.18 mg organic Se/kg DM. Horses consuming 0.39 mg organic

Se/kg DM had significantly greater whole blood Se concentrations as compared to horses

consuming 0.29 mg inorganic Se/kg DM at d 28 (Calamari et al., 2009). The results of

the current study disagree with Janicki et al. (2001), who reported no difference in serum

Se concentrations at d 28 of supplementation in pregnant mares supplemented with 1 or 3

mg Se/d. The results of this study disagree with Brummer et al. (2013), who reported no

significant differences in whole blood Se concentrations in horses consuming 0.12 mg

Se/kg DM, 0.3 mg inorganic Se/kg DM, or 0.3 mg organic Se/kg DM at d 28 of repletion.

A possible explanation for the differences in results observed in the current study and that

of Brummer et al. (2013) is the horses in Brummer’s study were consuming 60% of NRC

requirements and mean whole blood Se was much higher (165.1 ng/mL) at the end of

depletion as compared to the horses used in the current study (109 ng Se/mL).







71


Richardson et al. (2006), who reported significantly higher plasma Se concentrations in

horses consuming 0.45 mg organic and inorganic Se/kg DM as compared to horses

consuming 0.15 mg Se/kg DM. Additionally, these results agree with Richardson et al.

(2003), who reported plasma Se concentrations were significantly greater at d 56 in

horses consuming 0.6 mg organic Se/kg DM as compared to horses consuming 0.15 mg

Se/kg DM. Data from this study also agree with Janicki et al. (2001), who reported

significantly greater serum Se concentrations at d 56 of supplementation in mares

supplemented with 3 mg organic Se/d as compared to mares consuming 3 mg inorganic

Se/d and 1 mg inorganic Se/d. The results of this study partially agree with that of

Shellow et al. (1985), who reported significantly higher whole blood Se concentrations at

d 56 in horses consuming the 0.11, 0.16, and 0.26 ppm Se as compared to horses

consuming 0.06 ppm Se. Shellow et al. (1985) also reported significantly greater whole

blood Se concentrations in horses consuming 0.16 and 0.26 ppm Se as compared to

horses consuming 0.11 ppm Se. Data from this study also partially agree with Calamari et

al. (2009), who reported at d 56, horses consuming 0.18 mg organic Se/kg DM, 0.29 mg

organic Se/kg DM, 0.39 mg organic Se/kg DM, and 0.29 mg inorganic Se/kg DM had

significantly greater whole blood Se concentrations as compared to horses consuming

0.085 mg Se/kg DM. Furthermore, the researchers reported horses consuming 0.29 mg

organic Se/kg DM and 0.39 mg organic Se/kg DM had significantly greater whole blood

Se concentrations as compared to horses 0.18 organic Se/kg DM. Horses consuming 0.39

organic Se/kg DM had significantly greater whole blood Se concentrations as compared

72

to horses consuming 0.29 mg inorganic Se/kg DM at d 56 (Calamari et al., 2009). The

results of this study disagree with Brummer et al. (2013), who reported no significant

differences in whole blood Se concentrations in horses consuming 0.12 mg Se/kg DM,

0.3 mg inorganic Se/kg DM, and 0.3 mg organic Se/kg DM at d 56.



A significant effect of treatment was observed on overall mean whole blood Se


SE3 (mean = 136.4 ng Se/mL) had greater (P = 0.001) whole blood Se concentrations as

compared to horses consuming SE1 (mean = 120.0 ng Se/mL).


Janicki et al. (2001), who reported significantly greater serum Se concentrations at d 84

of supplementation in mares supplemented with 3 mg organic Se/d as compared to mares

consuming 3 mg inorganic Se/d and 1 mg inorganic Se/d. The results of this study

partially agree with that of Shellow et al. (1985), who reported significantly higher whole

blood Se concentrations at d 84 in horses consuming 0.11, 0.16, and 0.26 ppm Se as

compared to horses consuming 0.06 ppm Se. Shellow et al. (1985) also reported

significantly greater whole blood Se concentrations in horses consuming 0.16 and 0.26

ppm Se as compared to horses consuming 0.11 ppm Se. Data from this study also

partially agree with Calamari et al. (2009), who reported at d 84, horses consuming 0.18

mg organic Se/kg DM, 0.29 mg organic Se/kg DM, 0.39 mg organic Se/kg DM, and 0.29

mg inorganic Se/kg DM had significantly greater whole blood Se concentrations as

74

compared to horses consuming 0.085 mg Se/kg DM. Furthermore, the researchers

reported horses consuming 0.29 mg organic Se/kg DM and 0.39 mg organic Se/kg DM

had significantly greater whole blood Se concentrations as compared to horses 0.29 mg

inorganic Se/kg DM at d 84 (Calamari et al., 2009). Once again, overall mean whole

blood Se concentrations at d 84 were much lower than expected, and led to an unplanned

additional blood draw 12 days later on d 96.

Upon analysis of the raw data at d 84, whole blood Se concentrations decreased in

all horses regardless of treatment, indicating that the means at d 84 were not statistical

outliers, but rather a “real” biological event. The biological explanation for the decrease

at d 84 is unknown. A possible explanation is the extent of depletion in all horses, and a

possible RBC lifecycle effecting Se incorporation, at d 84. Stowe (1998) reported the life

span of equine RBC about 80 to 90 d. However, Carter et al. (1974) reported the lifespan

of erythrocytes in light horses to be 145 to 165 d.





SE3 (mean = 151.0 ng Se/mL) had greater (P = 0.001) whole blood Se concentrations as

compared to horses consuming SE1 (mean = 132.0 ng Se/mL).

There are no studies reporting the effects of Se supplementations on whole blood

Se concentrations at d 96. As previously stated, the unexpectedly low values observed at

d 84 led to an additional blood sampling at d 96. Overall mean whole blood Se

76

concentrations appeared to return to expected values based on the regression curve

previously developed and data from previous published studies.





SE3 (mean = 149.6 ng Se/mL) had significantly greater (P < 0.001) whole blood Se



that of Janicki et al. (2001), who reported significantly greater serum Se concentrations at

d 84 of supplementation in mares supplemented with 3 mg organic Se/d as compared to

mares consuming 3 mg inorganic Se/d and 1 mg inorganic Se/d. Data from this study

partially agrees with Calamari et al. (2009), who reported at d 112, horses consuming

0.18 mg organic Se/kg DM, 0.29 mg organic Se/kg DM, 0.39 mg organic Se/kg DM, and

0.29 mg inorganic Se/kg DM had significantly greater whole blood Se concentrations as

compared to horses consuming 0.085 mg Se/kg DM. Furthermore, Calamari et al. (2009)

reported horses consuming 0.29 mg organic Se/kg DM and 0.39 mg organic Se/kg DM

had significantly greater whole blood Se concentrations as compared to horses 0.18 mg

organic Se/kg DM, 0.29 mg inorganic Se/kg DM at d 84 .

Although Brummer et al. (2013) did not analyze whole blood Se concentrations

on d 112 as in the current study, the researchers reported significantly greater whole

78

blood Se concentrations at d 154 and 189 in horses consuming 0.3 mg organic and

inorganic Se/kg DM as compared to horses consuming 0.12 mg Se/kg DM.

Depletion Phase Erythrocyte Glutathione Peroxidase Activity Regression

At initiation of the depletion phase (d 0), overall mean erythrocyte (RBC) GSH-

Px activity were 42.33 ± 7.07 mU/mg Hb. Overall RBC GSH-Px activity in horses

consuming 23% of NRC Se recommendations depleted had large variation. Due to the

large variation, a non-linear regression equation could not be developed. Individual RBC

GSH-Px activities during the depletion phase can be observed in Figure B-1 in the

Appendix.

There are no published studies reporting the regression of a Se depletion phase.

However, data for Se depletion in horses has been reported. Brummer et al. (2013)

reported horses fed 60% of the NRC recommendation of Se for 196 d had significantly

lower whole blood GSH-Px activity at d 140 and 196 as compared to horses fed 0.12 mg

Se/kg DM. Furthermore, Brummer et al. (2013) reported significantly lower whole blood

GSH-Px activity in horses fed 0.06 mg Se/kg DM at d 84, 140, 168, and 196 as compared

to d 0. Whole blood GSH-Px activity concentrations in horses receiving 0.06 mg Se/kg

DM was significantly lower at d 168 and 196 as compared to d 84 and 140. However, the

authors reported no significant differences in whole blood GSH-Px activity between d

168 and 196. In the current study, overall mean RBC GSH-Px activity at the initiation of

depletion were 42.33 mU/mg Hb, as compared to Brummer et al. (2013), who reported

overall mean whole blood GSH-Px activity of 64.5 mU/mg Hb. In addition, at the end of

the depletion phase (d 112) in the current study, overall mean whole blood GSH-Px

79

activity in horses consuming 23% of NRC recommendations were 27.38 mU/mg Hb.

Brummer et al. (2013) reported whole blood GSH-Px activity in horses consuming 60%

of NRC recommendations of 52.7 mU/mg Hb at d 140, 46.7 mU/mg Hb at d 168, and

43.1 mU/mg Hb at d 196.

Repletion Phase Erythrocyte Glutathione Peroxidase Activity Regression

At the initiation of repletion phase (d 112 of depletion), horses were stratified

according to whole blood Se concentrations at d 84 of depletion, and assigned to 1 of 2

Se supplement treatments. Horses assigned to SE1 had overall mean RBC GSH-Px

activity of 27.28 ± 6.45 mU/mg Hb. Horses assigned to SE3 had overall mean whole

blood Se concentrations of 27.48 ± 4.12 mU/mg Hb.

Non-linear regression equations could not be developed due to variation within

sample. Individual RBC GSH-Px activities during repletion phase can be observed in

Appendix Figure B-2.

There are no studies reporting non-linear regression analysis of RBC GSH-Px

activity during a repletion phase. However, Calamari et al. (2009) reported linear

regressions of RBC GSH-Px activity {RBC GSH-Px activity, mU/L = 59.09 ± 9.70 x

time (d) + 12059 ± 2585} in horses consuming 0.18 mg Se yeast/kg DM, {RBC GSH-Px

activity, mU/L = 58.50 ± 7.87 x time (d) + 15149 ± 2555} in horses consuming 0.29 mg

Se yeast/kg DM, {RBC GSH-Px activity, mU/L = 63.88 ± 8.01 x time (d) + 10407 ±

2827} in horses consuming 0.39 mg Se yeast/kg DM, and {RBC GSH-Px activity, mU/L

= 90.74 ± 5.44 x time (d) + 3391 ± 1623} in horses consuming 0.29 mg Na selenite/kg

DM.

80

Erythrocyte Glutathione Peroxidase Activity in Horses Consuming 0.1 and 0.3 ppm

Selenium at d 0 of Repletion

There was no significant differences observed in overall mean RBC GSH-Px

activity in horses assigned to SE1 (mean = 27.28 mU/mg Hb) and SE3 (mean = 27.48

mU/mg Hb) at d 0 of repletion (P = 0.477; Figure 13).

Data for RBC GSH-Px activity at d 0 of repletion in the current study agree with

that of Brummer et al. (2013), who reported no differences in whole blood GSH-Px

activity in horses previously fed 0.06 mg Se/kg DM for 196 d at d 0 of repletion. The

results also agree with Calamari et al. (2009), who reported no differences in whole blood

GSH-Px activity in horses fed 0.085 mg Se/kg DM for 2 mo at the beginning of a

repletion phase. Richardson et al. (2003) reported no significant differences at the

beginning of a repletion phase in plasma and RBC GSH-Px activity in horses fed 0.15 mg

Se/kg DM for 28 d. Richardson et al. (2006) reported no significant differences at the

beginning of repletion in plasma, RBC, and muscle GSH-Px activity in horses fed 0.15

mg Se/kg DM for 28 d. Shellow et al. (1985) reported no significant differences in

plasma GSH-Px activity in horses fed 0.06 ppm Se for a minimum of 4 wk.



There was no significant effect of treatment observed on overall mean RBC GSH-

Px activity in horses consuming SE1 (mean = 30.38 mU/mg Hb) and SE3 (mean = 30.38


83


that of Shellow et al. (1985), who reported no significant differences in plasma GSH-Px

activity in horses consuming 0.06, 0.11, 0.16, and 0.26 ppm Se at d 14 of repletion.








activity in horses consuming 0.06 and 0.3 mg Se/kg DM at d 28 of repletion. These

results also agree with Calamari et al. (2009), who reported no differences in whole blood

GSH-Px activity in horses consuming 0.085, 0.18, 0.29, and 0.39 mg Se/kg DM at d 28

of repletion. In addition, data from the current study agrees with that of Shellow et al.

(1985), who reported no significant differences in plasma GSH-Px activity in horses fed

0.06, 0.11, 0.16, and 0.26 ppm Se at d 28 of repletion. The results of the current study

partially agrees with Richardson et al. (2003), who reported horses consuming 0.6 mg

organic Se/mg DM had significantly greater RBC GSH-Px activity at d 28 as compared

to horses consuming 0.15 mg organic Se/kg DM, and 0.6 inorganic Se/kg DM. Further,

Richardson et al. (2003) reported no significant differences at d 28 of repletion in plasma

RBC GSH-Px activity in horses 0.15 mg organic Se/kg DM, 0.6 mg organic and

inorganic Se/kg DM. Data from the current study also disagrees with that of Richardson

85

et al. (2006), who reported horses consuming 0.45 mg organic Se/kg DM tended to have

greater RBC GSH-Px activity at d 28 of repletion as compared to horses consuming 0.15

mg organic Se/kg DM and 0.45 mg inorganic Se/kg DM. Richardson et al. (2006)

reported no significant differences at d 28 of repletion in plasma GSH-Px activity in

horses fed 0.15 mg Se/kg DM, and 0.45 mg organic or inorganic Se/kg DM.








activity in horses consuming 0.06 and 0.3 mg Se/kg DM at d 56. The results from the

current study agree with that of Shellow et al. (1985), who reported no significant

differences in plasma GSH-Px activity in horses fed 0.06, 0.11, 0.16, and 0.26 ppm Se at

d 56. The results of the current study disagree with that of Calamari et al. (2009), who

reported significantly greater whole blood GSH-Px activity in horses consuming 0.18,

0.29, and 0.39 mg organic Se/kg DM and 0.39 mg inorganic Se/kg DM as compared

horses consuming 0.085 mg Se/kg DM at d 56 of repletion. In addition, Calamari et al.

(2009) reported horses consuming 0.39 mg inorganic Se/kg DM had significantly greater

whole blood GSH-Px activity as compared to horses consuming 0.29 mg organic Se/kg

DM. The results of the current study disagree with Richardson et al. (2003), who reported

87

horses consuming 0.6 inorganic Se/kg DM had significantly greater RBC GSH-Px

activity at d 56 of repletion as compared to horses consuming 0.15 mg organic Se/kg

DM, and tended (P = 0.057) to be greater as compared to horses consuming 0.6 mg

organic Se/mg DM. Further, Richardson et al. (2003) reported no significant differences

at d 56 of repletion in plasma RBC GSH-Px activity among horses consuming 0.15 mg

organic Se/kg DM, 0.6 mg organic or inorganic Se/kg DM. Data from the current study

also disagree with that of Richardson et al. (2006), who reported horses consuming 0.45

mg organic Se/kg DM tended to have greater RBC GSH-Px activity at d 56 of repletion

as compared to horses consuming 0.15 mg organic Se/kg DM and 0.45 mg inorganic

Se/kg DM. Richardson et al. (2006) reported no significant differences at d 56 of

repletion in plasma and muscle GSH-Px activity in horses fed 0.15 mg Se/kg DM, and

0.45 mg organic or inorganic Se/kg DM.







that of Shellow et al. (1985), who reported no significant differences in plasma GSH-Px

activity in horses fed 0.06, 0.11, 0.16, and 0.26 ppm Se at d 84. The results of the current

study disagree with that of Calamari et al. (2009), who reported significantly greater

whole blood GSH-Px activity in horses consuming 0.18, 0.29, and 0.39 mg organic Se/kg

89

DM and 0.39 mg inorganic Se/kg DM as compared horses consuming 0.085 mg Se/kg

DM at d 84 of repletion. In addition, Calamari et al. (2009) reported horses consuming

0.29, 0.39 mg organic Se/kg DM and 0.39 mg inorganic Se/kg DM had significantly

greater whole blood GSH-Px activity as compared to horses consuming 0.18 mg organic

Se/kg DM.






There are no published studies reporting the effects of Se supplementations on

RBC GSH-Px activity at d 96 of a repletion period. As previously stated, the

unexpectedly low values of whole blood Se concentrations observed at d 84 led to an

additional blood sampling at d 96. Overall mean RBC GSH-Px activity didn’t appear to

be affected by the apparent decline in whole blood Se concentrations at d 84.






Data for RBC GSH-Px activity at d 112 of repletion in the current study disagree

with that of Calamari et al. (2009), who reported significantly greater whole blood GSH-

92

Px activity in horses consuming 0.18, 0.29, and 0.39 mg organic Se/kg DM and 0.39 mg

inorganic Se/kg DM as compared to horses consuming 0.085 mg Se/kg DM at d 112 of

repletion. In addition, Calamari et al. (2009) reported horses consuming 0.29, 0.39 mg

organic Se/kg DM and 0.39 mg inorganic Se/kg DM had significantly greater whole

blood GSH-Px activity as compared to horses consuming 0.18 mg organic Se/kg DM.

Although Brummer et al. (2013) did not analyze whole blood GSH-Px activity on

d 112 as in the current study, the researchers reported significantly greater whole blood

GSH-Px activity in horses consuming 0.12 mg inorganic Se/kg DM, or 0.3 mg organic

and inorganic Se/kg DM as compared to horses consuming 0.085 organic Se/kg DM at d

154 of repletion. Additionally, the authors reported significantly greater whole blood

GSH-Px activity in horses consuming 0.3 mg inorganic Se/kg DM as compared to horses

consuming 0.12 mg inorganic Se/kg DM at d 154 of repletion. At d 189, Brummer et al.

(2013) reported horses consuming 0.12 mg inorganic Se/kg DM, or 0.3 mg organic and

inorganic Se/kg DM had significantly greater whole blood GSH-Px activity as compared

to horses consuming 0.085 mg organic Se/kg DM. Furthermore, at d 189, the authors

reported horses consuming 0.3 mg organic and inorganic Se/kg DM had significantly

greater whole blood GSH-Px activity as compared to horses consuming 0.12 mg

inorganic Se/kg DM.

Possible Explanation for Differences in Erythrocyte Glutathione Peroxidase Activity

between Studies

One possible explanation for the different results observed in the current study

and that of previous studies is the sample handling time and environmental temperature.

93

Koller et al. (1984) stated whole blood GSH-Px activity was less stable and reliable than

was whole blood Se concentrations. Hussein and Jones (1981) measured whole blood

GSH-Px activity in cattle, goats, and horses, and reported that both samples stored at

room temperature (20 °C), or in a refrigerator (5 °C), had considerably reduced enzyme

activity within 3 d, particularly in whole blood from horses. Jones (1985) reported that

whole blood GSH-Px activity was reduced by approximately 20% unless samples were

immediately frozen after blood draw. Abiaka et al. (2000) reported RBC GSH-Px activity

was stable in samples stored at -80 °C for approximately 2 yr. Additionally, the authors

reported that prior to freezing, plasma was separated and 0.9% NaCl solution was spun

with RBC at 2500 x g for 5 min using a non-temperature controlled centrifuge. In the

current study, the protocol for the assay kit (Bioxytech® GPx-340TM

; OxisResearchTM

,

Portland, OR) recommended centrifuging samples at 4 °C. However, the centrifuge used

in this study was not temperature controlled, therefore the possible change in temperature

could have increased the oxidation of GSH-Px. The resultant differing oxidation rates

could account for the differences in RBC GSH-Px activity observed in the current study

with data observed in previously mentioned studies.

94

Chapter V

CONCLUSIONS AND IMPLICATIONS

Results from this experiment allowed for the development of a depletion curve for

horses consuming 23% of the NRC Se recommendation for 112 d. The results also

indicate that horses may benefit from organic Se supplementation at levels higher than

those recommended by the NRC, during a 112-d repletion phase in previously depleted

horses. Variation in RBC GSH-Px activity suggests the importance of proper handling

and storage of GSH-Px samples to maintain the integrity of the blood samples. Whole

blood Se concentration data indicate that horses fed 0.3 ppm organic Se supplementation

will have higher whole blood Se concentrations over time as compared to horses

receiving 0.1 ppm organic Se supplementation.

The non-linear regression curve developed for horses consuming 23% of NRC Se

recommendation for 112 d was: {predicted whole blood Se concentration = 184.95 * (1 *

EXP (-0.005 * day))}. The non-linear regression curve for the change in whole blood Se

concentrations in horses consuming SE1, previously depleted to 108 ng Se/mL whole

blood was: {predicted change in whole blood Se concentration = 20.911 * (1 - EXP (-

0.062 * day))}. For horses consuming SE3, previously depleted to 109 ng Se/mL whole

blood, the non-linear regression curve was: {predicted change in whole blood Se

concentration = 38.249 * (1 - EXP (-0.070 * day))}.

95

Regression curves appeared to be greater in horses consuming SE3 as compared

to SE1. Horses consuming SE3 had greater whole blood Se concentrations at d 14, 28,

56, 84, 96, and 112 as compared to horses consuming SE1. Due to variation in RBC

GSH-Px activity, non-linear regression curves could not be developed for the depletion

phase, and each treatment during the repletion phase. No significant differences were

observed in RBC GSH-Px activity between treatments during repletion.

During the depletion phase, whole blood Se concentrations in this study mostly

agreed with that of Brummer et al. (2013). Whole blood Se concentrations during the

repletion phase mostly agreed with previously reported repletion studies. Data for RBC

GSH-Px activity from the current study both agreed and disagreed with previous studies.

Sample handling and storage may have affected the results of the RBC GSH-Px activity

assay. Data from the current study indicate that horses depleted to the extent of the

current study never reach their original values, even after organic Se supplementation for

112-d. Further research may be necessary to determine the time and dietary concentration

required to replenish Se stores in the body to adequate levels. In addition, further research

needs to address the economic and possible environmental impact of Se supplementation

in the horse industry.

96

LITERATURE CITED

Abiaka, C., F. Al-Awadi, and S. Olusi. 2000. Effect of prolonged storage on the activities

of superoxide dismutase, glutathione reductase, and glutathione peroxidase.

Clinical Chem. 46: 566-567.

Aleman, M. 2008. A review of equine muscle disorders. Neuromuscular Disorders.

18:277-287.

Avellini, L., E. Chiaradia, and A. Gaiti. 1999. Effect of exercise training, selenium and

vitamin E on some free radical scavengers in horses (Equus caballus) Compar.

Biochem. Phys. 123:147-154.

Blackmore, D.J., C. Campbell, C. Dant, J.E. Holden, and J.E. Kent. 1982. Selenium

status of Thoroughbreds in the United Kingdom. Equine Vet. J. 14(2):139-143.

Brummer, M., J.E. Ringler, A.G. Parks, S. Hayes, A.A. Adams, D.W. Horohov, and L.M.

Lawrence. 2009. Selenium status and equine immune function. J. Equine Vet. Sci.

29:362-363.

Brummer, M., S. Hayes, S.M. McCown, A.A. Adams, D.W. Horohov, and L.M.

Lawrence. 2011a. Selenium depletion reduces vaccination response in horses. J.

Equine Vet. Sci. 31:266.

Brummer, M., S. Hayes, J.E. Earing, S.M. McCown, and L.M. Lawrence. 2011b. Effect

of selenium depletion on oxidative stress in mature horses. J. Equine Vet. Sci. 31:

257.

Brummer, M., S. Hayes, K.A. Dawson, and L.M. Lawrence. 2013. Measures of

antioxidant status of the horse in response to selenium depletion and repletion. J.

Anim. Sci. 91:2158-2168.

Calamari, L., A. Ferrari, and G. Bertin. 2009. Effect of selenium source and dose on

selenium status of mature horses. J. Anim. Sci. 87: 167-178.

Calamari, L., F. Abeni, and G. Bertin. 2010. Metabolic and hematological profiles in

mature horses supplemented with different selenium sources and doses. J. Anim.

Sci. 88: 650-659.

97

Carmel, D.K., M.V. Crisman, W.B. Ley, M.H. Irby, and G.H. Edwards. 1989. A survey

of whole blood selenium concentrations of horses in Maryland. Proc. 11th

Eq.

Nutri. Phys. Sym. Stillwater, OK.

Carter, E.I., V.E. Valli, B.J. McSherry, F.J. Milne, G.A. Robinson, and J.H. Lumsden.

1974. The kinetics of hematopoiesis in the light horse I. the lifespan of peripheral

blood cells in the normal horse. Can. J. Comp. Med. 38: 303-313.

Charlton, S.J. and W.N. Ewing. 2007. The Minerals Directory. Context Products,

Nottingham, UK.

Chavez, E.R. 1979. Effect of dietary selenium depletion and repletion on plasma

glutathione peroxidase activity and selenium concentration in blood and body

tissue of growing pigs. Can. J. Anim. Sci. 59: 761-771.

Chiaradia, E., L. Avellini, F. Rueca, A. Spaterna, F. Porciello, M.T. Antonioni, and A.

Gaiti. 1998. Physical exercise, oxidative stress and muscle damage in racehorses.

Compar. Biochem. Phys. 119:833-836.

Daniels, L.A. 1996. Selenium metabolism and bioavailability. Bio. Trace. Element Res.

54: 185-199.

Desta, B., G. Maldonado, H. Reid, B. Puschner, J. Maxwell, A. Agasan, L. Humphreys,

and T. Holt. 2011. Acute selenium toxicosis in polo ponies. J. Vet. Diagn. Invest.

23(3): 623-628.

Dunnett, C.E., and M. Dunnett. 2008. Organic selenium and the exercising horses. In: 4th

European Workshop on Equine Nutri., Forssa, Finland. p. 33. (Nutrition of the

exercising horse. Markku T. Saatamoinen. P. 255)

Equine Nutrition. 2005. Selenium sources. J. Equine Vet. Sci. 25(1):35-36.

EXRX. Selenium. 2013. Accessed April 19 2013. http://www.exrx.net.nutrition/

antioxidants/selenium.html

Greiwe-Crandell, K.M., D.S. Kronfeld, G.A. Morrow, and W. Tiegs. 1993. Phosphorus

and selenium depletion in thoroughbred mares and weanlings. Proc. 13th

Equine

Nutri. Phys. Sym. Gainesville, FL.

Hussein, K.S., and B.E. Jones. 1981. Effects of different anticoagulants on determination

of erythrocyte glutathione peroxidase. Acta Vet. Scand. 22: 472-479.

98

Janicki, K.M., L.M. Lawrence, T. Barnes, and C.J. Stine. 2001. The effect of dietary

selenium source and level on selenium concentration, glutathione peroxidase

activity, and influenza titers in broodmares and their foals. Proc. 17th

Equine

Nutri. Phys. Sym. Lexington, KY.

Jones, D.G. 1985. Stability and storage characteristics of enzymes in cattle blood. Res.

Vet. Sci. 38: 301-306.

Karren, B.J., J.F. Thorson, C.A. Cavinder, C.J. Hammer, and J.A. Coverdale. 2010.

Effect of selenium supplementation and plane of nutrition on mares and their

foals: selenium concentrations and glutathione peroxidase. J. Anim. Sci. 88:991-

997.

Knight, D.A. and W.J. Tyznik. 1990. The effect of dietary selenium on humoral

immunocompetence of ponies. J. Anim. Sci. 68:1311-1317.

Koller, L.D., P.J. South, J.H. Exon, G.A. Whitbeck, and J. Mass. 1984. Comparison of

selenium levels and glutathione peroxidase activity in bovine whole blood. Can. J.

Comp. Med. 48: 431-433.

Koller, L.D., and J.H. Exon. 1986. The two faces of selenium- deficiency and toxicity-

are similar in animals and man. Can. J. Vet. Res. 50:297-306.

Lescure, A., M. Rederstorff, A. Krol, P. Guicheney, and V. Allamand. 2009.

Selenoprotein function and muscle disease. Biochimica et Biophysica Acta

(BBA)-General Subjects, 1790(11): 1569-1574.

Lewis, L.D. 1995. Feeding and care of the horse. 2nd

ed. Williams and Wilkins.

Baltimore, MD.

Ludvikova, E., L. Pavlata, M. Vyskocil, and P. Jahn. 2005. Selenium status of horses in

Czech Republic. ACTA Vet. BRNO. 74: 369-375.

Mayer, G. 2009. Immunoglobulins- Structure and function. Microbiology and

Immunology. http://pathmicro.med.sc.edu/mayer/igstruct2000.htm

Montgomery, J.B., J.J. Wichtel, M.G. Wichtel, M.A. McNiven, J.T. McClure, F.

Markham, and D.W. Horohov. 2012a. Effects of selenium source on measures of

selenium status and immune function in horses. Canadian J. Vet. Res. 76: 281-

291.

99

Montgomery, J.B., J.J. Wichtel, M.G. Wichtel, M.A. McNiven, J.T. McClure, F.

Markham, and D.W. Horohov. 2012b. The effects of selenium source on measure

of selenium status of mares and selenium status and immune function of their

foals. J. Equine Vet. Sci. 32:352-359.

Nicholson, J.W., R.E. McQueen, and R.S. Bush. 1991a. Response of growing cattle to

supplementation with organically bound or inorganic sources of selenium or yeast

cultures. Can. J. Anim. Sci. 71: 803-811.

Nicholson, J.W., J.G. Allen, and R.S. Bush. 1991b. Comparisons of responses in whole

blood and plasma selenium levels during selenium depletion and repletion of

growing cattle. Can. J. Anim. Sci. 71: 925-929.

NRC. 1983. Selenium in Nutrition, Revised Ed. Natl. Acad. Press, Washington, D.C.

NRC. 1989. Nutrient Requirements of Horses. 5th

rev. ed. Natl. Acad. Press, Washington,

DC.

NRC. 2007. Nutrient requirements of horses. 6th

rev. ed. Natl Acad. Press, Washington,

DC.

NIH. Selenium. 2013. Accessed August 14 2013. http://ods.od.nih.gov/factsheets/

Selenium-HealthProfessional/

Oldfield, J. E. 1998. Environmental implications of uses of selenium with animals.

Environmetal Chemistry of Selenium, 129-142.

Pagan, J.D., P. Karnezos, M.A.P. Kennedy, T. Currier, and K.E. Hoekstra. 2007. Effect

of selenium source of selenium digestibility and retention in exercised

thoroughbreds. Proc. 16th

Equine Nutri. Phys. Soc., Raleigh, N.C.

Podoll, K.L., J.B. Bernard, D.E. Ullrey, S.R. DeBar, P.K. Ku, and W.T. Magee. 1992.

Dietary selenate versus selenite for cattle, sheep, and horses. J. Anim. Sci. 70:

1965-1970.

Reddy, C.C. and E.J. Massaro. 1983. Biochemistry of selenium: a brief overview.

Fund. App. Tox. 3: 431-436.

Richardson, S.M., P.D. Siciliano, T.E. Engle, C.K. Larson, and T.L. Ward. 2006. Effect

of selenium supplementation and source on the selenium status of horses. J.

Anim. Sci. 84: 1742-1748.

100

Richardson, S.M., P.D. Siciliano, T.E. Engle, and T.L. Ward. 2003. Effect of selenium

supplementation and source (organic vs. inorganic) on selenium status on horses.

Proc. 18th

Equine Nutri. Phys. Sym. East Lansing, MI.

Shelle, J.E., W.D. VanHuss, J.S. Rook, and D.E. Ullrey. 1985. Relationship between

selenium and vitamin E nutrition and exercise in horses. 9th

Proc. Eq. Nutri. Phys.

Sym. East Lansing, MI.

Shellow, J.S., S.G. Jackson, J.P. Baker, and A.H. Cantor. 1985. The influence of dietary

selenium levels on blood levels of selenium and glutathione peroxidase activity in

the horse. J. Anim. Sci. 61: 590-594.

Sors, T.G., D.R. Ellis, and D.E. Salt. 2005. Selenium uptake, translocation, assimilation

and metabolic fate in plants. Photosynthesis Res. 86: 373-389.

Stadtman, T.C. 1983. New biologic functions- Selenium-dependent nucleic acids and

proteins. Fund. App. Tox. 3: 420-423.

Stowe, H.D. 1967. Serum selenium and related parameters of naturally and

experimentally fed horses. J. Nutri. 93: 60-64.

Stowe, H.D. and T.H. Herdt. 1992. Clinical assessment of selenium status of livestock. J.

Anim. Sci. 70:3928-3933.

Stowe, H.D. 1998. Selenium supplementation for horse feed. Nottingham Univ. Press,

Nottingham, UK.

Ullrey, D.E. 1987. Biochemical and physiological indicators of selenium status in

animals. J. Anim. Sci. 65: 1712-1726.

Ullrey, D.E. 1992. Basis for regulation of selenium supplements in animal diets. J. Anim.

Sci. 70:3922-3927.

White, S. 2010. Dietary selenium and prolonged exercise alter gene expression and

activity of antioxidant enzymes in equine skeletal muscle. MS Thesis. Univ.

Florida, Gainesville.

White, S., L.K. Warren, and J. Bobel. 2011. Effects of dietary selenium supplementation

and exercise on antioxidant enzyme activity in equine blood and skeletal muscle.

J. Equine Vet. Sci. 31:266-267.

Wright, P.L. and M.C. Bell. 1966. Comparative metabolism of selenium and tellurium in

sheep and swine. Am. J. Physiol. 211:6-10.

101

APPENDIX FIGURES A

WHOLE BLOOD SELENIUM

CONCENTRATIONS GRAPHS

105

APPENDIX FIGURES B

ERYTHROCYTE GLUTATHIONE PEROXDIASE

ACITIVITY GRAPHS

108

APPENDIX TABLES A

109

Table A-1. Individual Whole Blood Selenium Concentrations (ng/mL)

Depletion phase (d)

Horse

0 28 56 84 112

1

187 158 139 127 113

2

175 138 120 104 89

3

191 153 133 122 106

4

193 171 145 139 122

5

199 172 140 128 111

6

194 161 150 148 122

7

188 152 129 125 103

8

187 161 137 132 114

9

181 162 144 137 116

10

179 156 126 100 94

Repletion phase (d)

Horse Supplement (ppm)

14 28 56 84 96 112

1 0.1

117 134 143 126 138 135

2 0.1

100 114 121 111 125 119

3 0.1

115 130 134 118 129 126

4 0.1

131 141 142 128 138 137

5 0.1

115 128 135 117 130 124

6 0.3

139 158 162 145 165 161

7 0.3

123 144 148 132 146 145

8 0.3

137 150 159 135 150 147

9 0.3

134 146 155 139 149 152

10 0.3

116 137 142 131 145 143

110

Table A-2. Individual Erythrocyte Glutathione Peroxidase Activity (mU/mg Hb)

Depletion phase (d)

Horse 0 28 56 84 112

1

34.82 42.35 34.65 48.32 24.28

2

48.72 34.47 50.75 34.76 22.65

3

39.86 38.87 42.87 40.84 29.14

4

47.80 40.67 56.14 46.93 37.77

5

34.59 33.31 47.39 32.44 22.54

6

40.56 33.37 43.34 46.41 31.63

7

40.32 43.92 46.29 40.32 23.29

8

57.41 45.60 41.02 26.88 24.97

9

37.77 45.65 44.32 37.83 32.21

10

41.48 46.29 35.69 26.19 25.32

Repletion phase (d)

Horse Supplement (ppm) 14 28 56 84 96 112

1 0.1

32.04 28.10 20.28 22.25 22.48 33.54

2 0.1

21.03 30.13 23.06 40.55 30.24 36.62

3 0.1

24.68 24.04 25.09 26.01 25.20 44.96

4 0.1

39.34 35.28 38.82 38.12 37.89 35.28

5 0.1

34.82 26.48 22.71 32.44 27.29 30.36

6 0.3

26.65 28.16 32.91 31.69 21.55 26.24

7 0.3

33.89 34.94 31.23 30.13 37.02 32.85

8 0.3

33.89 33.66 32.79 21.49 25.78 38.18

9 0.3

34.30 35.22 24.10 36.33 26.30 28.21

10 0.3

23.17 16.74 18.89 17.03 26.19 35.69

111

Table A-3. Individual Body Weights (kg)

Depletion

Horse 0 14 28 56 84 112

1

570 550.7 540.7 538 552 547

2

494.2 478.7 475.7 466 481 491

3

509.8 487.6 486.8 482 492 493

4

484.2 441.4 433.7 433 443 445

5

601.2 522.3 507.4 507 517 519

6

506.5 491.3 495.3 488 486 490

7

565.4 539 540.7 540 549 540

8

604.4 549.6 546.6 540 539 536

9

569.5 559.1 540.1 528 544 544

10

537.8 529 519.4 514 538 534

Repletion

Horse Supplement (ppm) 14 28 56 84 112

1 0.1

549 543 541 537 547.5

2 0.1

486 481 491 481 491

3 0.1

492 488 487 481 488.3

4 0.1

438 435 435 434 435.3

5 0.1

519 514 527 526 537

6 0.3

490 486 474 478 478.6

7 0.3

540 527 529 528 529.2

8 0.3

527 527 519 521 523.7

9 0.3

533 529 533 532 534

10 0.3

529 525 527 519 532.1

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