Steadfast Equine Whitepaper

WHITEPAPER

TABLE OF CONTENTS

I. Executive Summary����������������������������������������������������������������������������������������������������������������������������������3
II. Ingredients – Description, Inclusion Rationale, Mechanism of Action�����������������������������������������3

a. Biotin�������������������������������������������������������������������������������������������������������������������������������������������������3
b. Methionine���������������������������������������������������������������������������������������������������������������������������������������3
c. Zinc����������������������������������������������������������������������������������������������������������������������������������������������������4
d. Copper�����������������������������������������������������������������������������������������������������������������������������������������������4
e. Manganese��������������������������������������������������������������������������������������������������������������������������������������4
f. TêlaFIRM®�����������������������������������������������������������������������������������������������������������������������������������������4
g. Selenium�������������������������������������������������������������������������������������������������������������������������������������������4
h. Ascorbic Acid����������������������������������������������������������������������������������������������������������������������������������4
i. Vitamin D��������������������������������������������������������������������������������������������������������������������������������������������5
j. Calcium & Phosphorus��������������������������������������������������������������������������������������������������������������������5
k. Glucosamine, Chondroitin & Hyaluronic Acid���������������������������������������������������������������������������5
III. Closing������������������������������������������������������������������������������������������������������������������������������������������������������6
IV. References������������������������������������������������������������������������������������������������������������������������������������������� 6-7

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Do not photocopy without the written permission of Altera International, Ltd. For discussion purposes only.
References are for informational purposes. Altera does not necessarily agree with references cited or the

2 conclusions of the researchers.

I. EXECUTIVE SUMMARY 3

STEADFAST® Equine, a product of Arenus, is a unique and comprehensive supplement
for total structural health. It is comprised of the nutritional components necessary
to support the health and growth of hooves, bones, joints, tendons and ligaments in
growing and performance horses. STEADFAST® Equine contains the keystones for
production and maintenance of healthy hooves, biotin and methionine (as methionine
hydroxyl analogue). STEADFAST® Equine also provides a natural source of supportive
nutrients for healthy joint function from Natural Eggshell Membrane (NEM®), including
glucosamine, chondroitin, and hyaluronic acid. In addition, STEADFAST® Equine utilizes
an advanced micronutrient complex (TêlaFIRM®) that contains organic sources of
zinc, copper and manganese with improved bioavailability over standard trace mineral
formulations [27]. Moreover, TêlaFIRM® also provides organic selenium (Se) source
shown to have higher bioavailability and tissue retention [29]. Providing trace nutrients
in these highly bioavailable forms is important since most commercial feeds and
supplements utilize forms shown to be less stable through the GI tract and potentially
more susceptible to antagonisms. This decreased stability and bioavailability may result
in inadequate levels of micronutrients to meet the increased metabolic requirements
of growing and performance horses. STEADFAST® Equine also provides calcium (Ca),
phosphorous (P), vitamin C and vitamin D, all which play important roles in the production
and maintenance of healthy bone and cartilage. Flaxseed oil, a good source of essential
omega-3 fatty acids known to reduce inflammation, is provided in a stabilized form
using the antioxidant, Natuguard™. STEADFAST® Equine supplies these key nutritional
components in a highly palatable alfalfa/molasses mix.

II. INGREDIENTS

DESCRIPTION, INCLUSION RATIONALE,
AND MECHANISM OF ACTION

a. Biotin

STEADFAST® Equine provides 20 mg of biotin, a water-soluble B vitamin involved in the
formation of all connective tissue, and especially important in the maintenance of high
quality hoof health. Several studies have demonstrated that the daily supplementation
of a least 15 mg of dietary biotin consistently improves hoof growth and strength in
horses when fed over a period of several months [3,9,22]. Biotin acts by increasing the
rate of keratin formation in the hoof wall [22]. Keratin proteins provide the structural
basis for the unique properties of the hoof wall and its protective function against use
and the environment. The hoof wall is produced through a process of differentiation of
living epidermal cells into cornified, structurally stable cells connected by an intercellular
cementing substance in a process termed keratinization. Biotin is an essential enzyme
in the metabolism of keratinizing epidermal cells and a deficiency leads to cracked and
brittle hooves with little regeneration and slow growth.

b. Methionine

The strength of the keratin formed in the hoof is determined by the cross-linking of
sulphur-containing amino acids provided by and between cystine (Cys) residues [19].
Methionine is an essential sulfur-containing amino acid which is critical for the production
of cystine. Methionine also acts as an antioxidant to reduce inflammation, as the sulfur
it supplies inactivates free radicals and increases lecithin production, as well as the
synthesis of phosphatidylcholine and other phospholipids required for cartilage, ligament
and tendon growth. In addition, methionine provides sulfur to promote healthy skin and
coat. STEADFAST® Equine provides a supplemental source of methionine as methionine
hydroxyl analogue. Methionine hydroxyl analogue differs from dl, methionine in its
chemistry and has been demonstrated to differ in its absorption and utilization within
the body. Advantages of methionine hydroxyl analogue as compared to dl, methionine
include more rapid absorption and anti-microbial activity against certain undesirable
species of bacteria and mold.

c. Zinc

Trace minerals and vitamins play important roles in the production and maintenance of healthy keratinized tissues.
Zinc (Zn) has a role in three key functions in the keratinization process; catalytic, structural and regulatory [4]. Zinc
serves as an enzyme activator in the differentiation process of keratinocytes [8] and is involved in the formation of
structural proteins during the keratinization process [4]. Zinc also regulates calmodulin and thus the calcium binding
necessary for the final step of the differentiation process. Organic zinc also exhibits anti-inflammatory activity in
combination with copper by prevention of lipid peroxidation [17, 19].

d. Copper

Copper (Cu) is instrumental in the activation of enzymes throughout metabolism and is essential for the transport
of iron for hemoglobin synthesis. Copper activates the enzyme, thiol oxidase, which is responsible for the
formation of disulfide bonds between the cystine residues of the keratin that comprises the hoof wall [21].
Copper is also an integral component of the enzyme responsible for the cross-linking of collagen molecules in the
maturation of collagen [25].

e. Manganese

Similar to zinc and copper, manganese (Mn) has anti-inflammatory activities through activation of manganese
superoxide dismutase (Mn SOD) and the removal of free radicals. It is proposed that this process protects the
lipids involved in cementing together mature keratinocytes and thus improves hoof quality and growth [18]. In
addition, manganese plays a central role in the synthesis of glycosaminoglycans and glycoproteins, which are
important constituents of cartilage and bone. Manganese promotes the formation of normal cartilage and bone
through the activation of enzymes needed for synthesis of the chondroitin-sulfate side chains of proteoglycan
molecules [12].

f. TêlaFIRM®

Increasing the bioavailability of trace minerals improves their utilization and thus can help improve the integrity
of bone, cartilage, tendons, ligaments and hooves [1]. STEADFAST® Equine contains organic sources of zinc,
copper and manganese in the advanced micronutrient complex, TêlaFIRM®. The form of these minerals found
in TêlaFIRM® has been shown to have greater stability and absorption than other forms commonly found in
commercial feeds and supplements.

g. Selenium

As in the case of zinc, copper, and manganese, selenium (Se) has anti-inflammatory activity as a constituent
of the enzyme glutathione peroxidase, which protects both intra- and extra-cellular lipid membranes against
oxidative damage. In this manner, selenium may contribute to the protection and maintenance of the physiological
function of the lipid-rich intercellular cementing substance that is responsible for hoof integrity. As an antioxidant,
selenium works with vitamin E to maintain muscle health and function. A deficiency in selenium, as may occur
in the Eastern U.S. and Canada, can result in muscular dystrophy in foals, or increase the incidence of “tying
up” in working horses [14]. Oral supplementation of selenium at a level of approximately 1 mg per day has been
demonstrated to increase blood selenium concentrations above levels associated with myodegeneration in horses
and foals [16]. Alternatively, excess selenium is toxic resulting in blindness, muscle paralysis, mane and tail
alopecia, as well as hoof sloughing [26]. TêlaFIRM® in STEADFAST® Equine supplies 1.75 µg of the trace mineral
as an organic source having improved bioavailability and higher retention in tissues [29].

h. Ascorbic Acid

Ascorbic acid (vitamin C) is an important cofactor for collagen synthesis [10] and bone cell proliferation [30].
Ascorbic acid is required for the hydroxylation of proline and lysine to hydroxyproline and hydroxylysine, the
structural backbones of collagen [10]. In addition, ascorbic acid stimulates bone cell growth and strength by
increasing chondrocyte production [30], osteoblast differentiation, and bone mineralization by increased alkaline
phosphatase activity [6] Although horses are able to synthesize some ascorbic acid in the liver, adequate levels are
essential to maintain healthy ligaments, bone, cartilage and teeth [15]. Ascorbic acid is also a strong antioxidant
that may be involved in improving both cardiac and immune system health [11]. Ascorbic acid is supplied by
STEADFAST® Equine at a rate of 1000 mg/day.

4

i. Vitamin D

Vitamin D (Calciferol) is one of the most important biological regulators of calcium
metabolism and as such, is critical for normal bone growth and development. The
biologically active form of vitamin D, 1, 25(OH)2D3 or vitamin D3 facilitates the
absorption of calcium+2 and phosphorus from the intestine and utilization of these
minerals for bone formation. Vitamin D3 is required for the control of calcium+2
reabsorption, absorption and mobilization/accretion from bones [20]. While horses
normally obtain adequate amounts of vitamin D from exposure to sunlight and from
sun-cured forages, the increased stabilization and reliance of pelleted complete feeds in
today’s performance horses may lead to a deficiency leading to bone that is weak, soft
and easily deformed. Vitamin D3 is supplied in STEADFAST® Equine at a level of 1000
I.U. per day.

j. Calcium & Phosphorus

In addition to trace minerals and vitamins, STEADFAST® Equine provides both calcium
and phosphorus in the form of dicalcium phosphate. Calcium and phosphorus are the
most abundant elements in the body, the majority of both being present in the bone
as hydroxyapatite [24]. Bone, which has a Ca:P ratio of 2:1, is intimately associated
with collagen formation. Calcium is also necessary for blood clotting, nerve impulse
transmission, muscle contraction, enzyme activation and hormone secretion. Phosphorus
is part of many structural and functional compounds in the body such as phospholipids
and phosphoproteins. Calcium and phosphorus requirements are increased in young,
growing horses to meet the needs of skeletal development. Calcium and phosphorus
needs are also increased in the pregnant mare during late pregnancy for bone formation
in the developing fetus and during lactation to support the secretion of milk.

k. Glucosamine, Chondroitin & Hyaluronic Acid

STEADFAST® Equine is unique in that it not only provides a complete source of the
essential nutrients necessary to support the growth and maintenance of the structural
components of the leg, but maintains and promotes healthy joints. STEADFAST®
Equine provides glucosamine, chondroitin and hyaluronic acid, critical for the production
of glycosaminoglycans (GAGs), the biologically-produced building blocks of cartilage,
connective tissue and lubricating joint fluids.
Glucosamine is the main component of GAGs and is required for the production of
chondroitin sulfate, the primary GAG found in joint cartilage and connective tissue,
as well as hyaluronic acid, a natural joint lubricant. Chondroitin sulfate not only serves
in a synthetic capacity, but acts to inhibit enzymes that destroy cartilage, as well.
Glucosamine, chondroitin sulfate and hyaluronic acid supplementation has been reported
to relieve symptoms of joint inflammation and arthritis [5]. However, determination of
the dosage of each component necessary for optimal efficacy is unclear. STEADFAST®
Equine provides all three key components of joint health in a natural biologic ratio from a
single source, Natural Eggshell Membrane (NEM®). NEM® is a single source supplement
containing a unique matrix of all-natural nutrients including glucosamine, chondroitin
sulfate, and hyaluronic acid in the appropriate biologic ratio [7]. Clinical studies with
NEM® have demonstrated a significant reduction in pain and improved range of motion in
the joints of patients suffering from arthritis and a variety of joint and connective tissue
injuries [13]. Dogs that demonstrated suboptimal joint function were fed STEADFAST®
Canine over a six-week clinical study further demonstrated rapid improvement in pain
and joint function [23]. Another study saw a significant increase in tarsal flexion in
osteoarthritic cranes fed STEADFAST® [2]. Rats with osteoarthritis in one hindlimb were
able to bear more weight on the affected limb 14 days after beginning supplementation
with STEADFAST® and both rats and horses fed STEADFAST® saw a decrease in the
cartilage degradation biomarker CTX-II, suggesting the chondromodulating effects of
STEADFAST® [28]. Daily supplementation with STEADFAST® provides 3000 mg of
NEM®.

5

III. CLOSING

STEADFAST® Equine is formulated in a highly palatable mixture of flaxseed oil, a good source of essential
omega-3 fatty acids known to reduce inflammation, alfalfa and molasses mix. Daily supplementation with
STEADFAST® Equine supplies all of the nutritional components necessary for supporting the growth and health
of all structural components (i.e. hoof, bones, cartilage, ligaments and tendons in addition to providing anti-
inflammatory activity for improved joint health and flexibility of performance horses.

IV. REFERENCES

[1] Ballantine HT, Socha MT, Tomlinson DJ, Johnson AB, Fielding AS, Shearer JK, Amstel SR. Effects of feeding complexed zinc,
manganese, copper and cobalt on hoof integrity, reproduction and lactation performances. Prof Anim Sci. 2002; 18: 211-218.

[2] Bauer KL, Dierenfeld ES, and Hartup BK: Evaluation of a nutraceutical joint supplement in cranes. Proc N Amer Crane Workshop.
2014; 12: 27-32.

[3] Buffa EA, Van Den Berg SS, Verstraete FJ, Swart NG. Effect of dietary biotin supplement on equine hoof horn growth rate and
hardness. Equine Vet J. 1992; 24: 472-474.

[4] Cousins RJ. Present Knowledge in Nutrition, 7th edition. E.E. Zeigler & L.J. Filer, Jr., ed. ILSI Press, Washington, D.C. 1996; 293-
306.

[5] Deal CL, Moskowitz RW. Nutraceuticals as therapeutic agents in osteoarthritis. The role of glucosamine, chondroitin sulfate, and
collagen hydrolysate. Rheum Dis Clin North Am. 1999; 25(2): 379-395.

[6] Denis I, Pointillart A, Lieberherr M. Cell stage-dependent effects of ascorbic acid on cultured porcine bone cells. Bone Miner. 1994;
15: 149-161.

[7] ESM Technologies. NEM® - 10 day results for joints. Accessed July 3, 2007. http://www.esmingredients.com/branded-ingredients/
nem/.

[8] Fraser RD, MacRae TP. The Mechanical Properties of Biological Materials. J.F. Vincent & D. Curry, ed. Cambridge University Press,
Cambridge, U.K. 1980; 211-246.

[9] Geyer H, Schulze J. The long-term influence of biotin supplementation on hoof horn quality in horses. Schweiz Arch Tierheilkd.
1994; 136(4): 137-149.

[10] Hausmann E. Cofactor requirements for the enzymatic hydroxylation of lysine in a polypeptide precursor of collagen. Biophys
Acta. 1967; 133: 591-593.

[11] Jacob RA, Sotooudeh G. Vitamin C function and status in chronic disease. Nutr Clin Care. 2002; 5: 66-74.
[12] Keen CL, Zidenberg-Charr S. Present Knowledge in Nutrition, 6th ed. M.L. Brown, ed. ILSI Press, Washington, D.C. 1990; 334-

343.
[13] Leu M, DeVore D. Clinical Summary: Reduction of joint and soft tissue pain with natural eggshell membrane compositions.

Accessed July 26, 2007. http://www.internetarthritiscenter.com/nem1.html.
[14] Löfstedt J. White muscle disease of foals. Vet Clins North Am Equine Pract. 1997; 13: 169-185.
[15] Lowe G, Woodward M, Rumley A, Morrison C, Tunstall-Pedoe H, Stephen K. Total tooth loss and prevalent cardiovascular disease

in men and women: possible roles of citrus fruit consumption, vitamin C, and inflammatory and thrombotic variables. J Clin
Epidmiol. 2003; 56: 694-700.
[16] Maylin GA, Rubin DS, Lein DH. Selenium and vitamin E in horses. Cornell Vet. 1980; 70: 272- 289.
[17] Mülling C. The use of nutritional factors in prevention of claw diseases - Biotin as an example for nutritional influences on
formation and quality of hoof horn. Proceedings of the 11th International Symposium on Disorders of the Ruminant Digit, Parma,
Italy. 2000; 78-80.
[18] Mülling C. Three-dimensional appearance of epidermal keratinocytes in different stages of differentiation revealed by cell
maceration and scanning electron microscope investigation. Folia Morphol. 2000; 59: 239-246.
[19] Mülling CK, Bragulla HH, Reese S, Budras KD, Steinberg W. How structures in bovine hoof epidermis are influenced by nutritional
factors. Anat Histol Embryol. 1999; 28(2): 103-108.
[20] Norman AW. Present Knowledge in Nutrition, 6th ed. M.L. Brown, ed. ILSI Press, Washington, D.C. 1990; 120-129.
[21] O’Dell BL. Present Knowledge in Nutrition, 6th ed. M.L. Brown, ed. ILSI Press, Washington, D.C. 1990; 261-267.
[22] Reilly JD, Cottrell DF, Martin RJ, Cuddeford DJ. Effect of supplementary dietary biotin on hoof growth and hoof growth rate in
ponies: a controlled trial. Equine Vet J Suppl. 1998; 26: 51-57.
[23] Ruff KJ, Kopp KJ, Von Behrens P, Lux M, Mahn M, Back M. Effectiveness of NEM® brand eggshell membrane in the treatment
of suboptimal joint function in dogs: a multicenter, randomized, double-blind, placebo-controlled study. J Vet Med: Research and
Reports. 2016; 7: 113-121.
[24] Schryver HF, Hintz HF. Current Therapy in Equine Medicine. D. Pedersen, ed. W.B. Saunders, Philadelphia, PA. 1987; 395-398.
[25] Schryver HF, Hintz HF. Current Therapy in Equine Medicine. Pedersen, ed. W.B. Saunders, Philadelphia, PA. 1987; 400-401.
[26] Schryver HF, Hintz HF. Current Therapy in Equine Medicine. D. Pedersen, ed. W.B. Saunders, Philadelphia, PA. 1987; 402-404.
[27] Vàzquez-Añón M, Peters T, Hampton T, McGrath J, Huedepohl B. Case study: Supplementation of chelated forms of zinc, copper,
and manganese to feedlot cattle with access to drinking water with high sulfate concentration. Prof Anim Sci. 2007; 23: 58-63.
[28] Wedekind KJ, Coverdale JA, Hampton, TR, Atwell CA, Sorbet RH, Lunnemann J, Harrell RJ, Greiner L, Keith NK, Evans JL, Zhao
J, Knight CD. Efficacy of an equine joint supplement, and the synergistic effect of its active ingredients (chelated trace minerals
and natural eggshell membrane), as demonstrated in equine, swine and an osteoarthritis rat model. Animal Physiology. 2015; 7:

6 13-27.

[29] Wilde D. Influence of macro and micro minerals in the peri-parturient period on fertility in dairy
cattle. Anim Reprod Sci. 2006; 96(3-4): 240-249.

[30] Wright GJ, Wei XQ, McDevitt CA, Lane B, Sokoloff L. Stimulation of matrix formation in rabbit
chondrocyte cultures by ascorbate. J Orthop Res. 1988; 6: 397-407.

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