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
ii
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
iv
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.
v
Approved:
__________________________________________ ____________
Chair, Thesis Committee Date
__________________________________________ ____________
Member, Thesis Committee Date
__________________________________________ ____________
Member, Thesis Committee Date
_________________________________________ ____________
Member, 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
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and
0.3 ppm Selenium at d 14 of Repletion 65
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and
0.3 ppm Selenium at d 28 of Repletion 67
viii
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and
0.3 ppm Selenium at d 56 of Repletion 69
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and
0.3 ppm Selenium at d 84 of Repletion 72
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and
0.3 ppm Selenium at d 96 of Repletion 74
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and
0.3 ppm Selenium at d 112 of Repletion 76
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
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming
0.1 and 0.3 ppm Selenium at d 14 of Repletion 80
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming
0.1 and 0.3 ppm Selenium at d 28 of Repletion 83
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming
0.1 and 0.3 ppm Selenium at d 56 of Repletion 85
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming
0.1 and 0.3 ppm Selenium at d 84 of Repletion 87
ix
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming
0.1 and 0.3 ppm Selenium at d 96 of Repletion 89
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming
0.1 and 0.3 ppm Selenium at d 112 of Repletion 89
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
7. Overall mean whole blood selenium concentrations at d 14 of
selenium repletion 66
xii
8. Overall mean whole blood selenium concentrations at d 28 of
selenium repletion 68
9. Overall mean whole blood selenium concentrations at d 56 of
selenium repletion 70
10. Overall mean whole blood selenium concentrations at d 84 of
selenium repletion 73
11. Overall mean whole blood selenium concentrations at d 96 of
selenium repletion 75
12. Overall mean whole blood selenium concentrations at d 112 of
selenium repletion 77
13. Overall mean erythrocyte glutathione peroxidase activity at d 0
of selenium repletion 81
14. Overall mean erythrocyte glutathione peroxidase activity at d 14
of selenium repletion 82
15. Overall mean erythrocyte glutathione peroxidase activity at d 28
of selenium repletion 84
16. Overall mean erythrocyte glutathione peroxidase activity at d 56
of selenium repletion 86
17. Overall mean erythrocyte glutathione peroxidase activity at d 84
of selenium repletion 88
18. Overall mean erythrocyte glutathione peroxidase activity at d 96
of selenium repletion 90
xiii
19. Overall mean erythrocyte glutathione peroxidase activity at d 112
of selenium repletion 91
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.
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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.
56
57
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.
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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.
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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).
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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.
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and 0.3 ppm Selenium
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
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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.
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and 0.3 ppm Selenium
at d 28 of Repletion
A significant effect of treatment was observed on overall whole blood Se
concentrations in horses consuming SE1 and SE3 at d 28 (Figure 8). Horses consuming
SE3 (mean = 147.0 ng Se/mL) had significantly greater (P = 0.007) whole blood Se
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
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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).
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and 0.3 ppm Selenium
at d 56 of Repletion
A significant effect of treatment was observed on overall whole blood Se
concentrations in horses consuming SE1 and SE3 at d 56 (Figure 9). Horses consuming
SE3 (mean = 153.2 ng Se/mL) had significantly greater (P = 0.004) whole blood Se
concentrations as compared to horses consuming SE1 (mean = 135.0 ng Se/mL).
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71
Data for whole blood Se concentrations at d 56 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 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.
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and 0.3 ppm Selenium
at d 84 of Repletion
A significant effect of treatment was observed on overall mean whole blood Se
concentrations in horses consuming SE1 and SE3 at d 84 (Figure 10). Horses consuming
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).
Data for whole blood Se concentrations at d 84 of the current study agree with
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
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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.
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and 0.3 ppm Selenium
at d 96 of Repletion
A significant effect of treatment was observed on overall whole blood Se
concentrations in horses consuming SE1 and SE3 at d 96 (Figure 11). Horses consuming
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
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concentrations appeared to return to expected values based on the regression curve
previously developed and data from previous published studies.
Whole Blood Selenium Concentrations in Horses Consuming 0.1 and 0.3 ppm Selenium
at d 112 of Repletion
A significant effect of treatment was observed on overall whole blood Se
concentrations in horses consuming SE1 and SE3 at d 112 (Figure 12). Horses consuming
SE3 (mean = 149.6 ng Se/mL) had significantly greater (P < 0.001) whole blood Se
concentrations as compared to horses consuming SE1 (mean = 128.2 ng Se/mL).
Data for whole blood Se concentrations at d 112 of the current study agree with
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
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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
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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.
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming 0.1 and 0.3 ppm
Selenium at d 14 of Repletion
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
mU/mg Hb) at d 14 of repletion (P = 0.500; Figure 14).
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Data for RBC GSH-Px activity at d 14 of repletion in the current study agree with
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.
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming 0.1 and 0.3 ppm
Selenium at d 28 of Repletion
There was no significant effect of treatment observed on overall mean RBC GSH-
Px activity in horses consuming SE1 (mean = 28.81 mU/mg Hb) and SE3 (mean = 29.74
mU/mg Hb) at d 28 of repletion (P = 0.411; Figure 15).
Data for RBC GSH-Px activity at d 28 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 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
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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.
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming 0.1 and 0.3 ppm
Selenium at d 56 of Repletion
There was no significant effect of treatment observed on overall mean RBC GSH-
Px activity in horses consuming SE1 (mean = 25.99 mU/mg Hb) and SE3 (mean = 27.98
mU/mg Hb) at d 56 of repletion (P = 0.328; Figure 16).
Data for RBC GSH-Px activity at d 56 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 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
86
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.
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming 0.1 and 0.3 ppm
Selenium at d 84 of Repletion
There was no significant effect of treatment observed on overall mean RBC GSH-
Px activity in horses consuming SE1 (mean = 31.88 mU/mg Hb) and SE3 (mean = 27.33
mU/mg Hb) at d 84 of repletion (P = 0.193; Figure 17).
Data for RBC GSH-Px activity at d 84 of repletion in 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 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
88
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.
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming 0.1 and 0.3 ppm
Selenium at d 96 of Repletion
There was no significant effect of treatment observed on overall mean RBC GSH-
Px activity in horses consuming SE1 (mean = 28.62 mU/mg Hb) and SE3 (mean = 27.37
mU/mg Hb) at d 96 of repletion (P = 0.371; Figure 18).
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.
Erythrocyte Glutathione Peroxidase Activity in Horses Consuming 0.1 and 0.3 ppm
Selenium at d 112 of Repletion
There was no significant effect of treatment observed on overall mean RBC GSH-
Px activity in horses consuming SE1 (mean = 36.15 mU/mg Hb) and SE3 (mean = 32.24
mU/mg Hb) at d 112 of repletion (P = 0.135; Figure 19).
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-
90
91
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
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101
APPENDIX FIGURES A
WHOLE BLOOD SELENIUM
CONCENTRATIONS GRAPHS
102
103
104
105
APPENDIX FIGURES B
ERYTHROCYTE GLUTATHIONE PEROXDIASE
ACITIVITY GRAPHS
106
107
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