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Apr 25

 Use of Nutrigenomics in Equine Research

  posted by helyn on 25.04.11 19:29 as Equine Science




Ronan F. Power, PhD

Author’s address: Center for Animal Nutrigenomics and Applied Animal Nutrition, 3031 Catnip
Hill Pike, Nicholasville, KY 40356; e-mail: rpower@alltech.com.

Take Home Message

DNA microarrays are capable of examining the gene expression profile of thousands of genes in
a single experiment. Nutrigenomics is one particular application of this technology where the
genome-wide impact of nutrients on the expression of genes is used to elucidate pathways
leading to disease states, improved health, or enhanced performance. The recent publication of
the horse genome sequence will make this powerful research tool more available to those
involved in equine science.

Introduction

The National Institute of Health (NIH) added the horse genome (Equus caballus) to the list of
mammals to undergo whole genome, high density sequencing in 2006.1 The purpose of the
project was to obtain a high-quality draft sequence of the Thoroughbred horse genome; the
animal behind a multi-billion dollar global industry. Gaining an understanding of the expression
of specific genes in equines offers researchers a unique insight into the molecular and biological
processes responsible for health and performance in the horse. The 2.7 Gigabase (Gb) horse
genome was sequenced in 2007 but the final report on assembly and comparative analysis did
not appear until late 2009.

The genome sequence for the horse comes from a blood sample taken from a single
Thoroughbred mare called Twilight; chosen because she had the least genetic diversity amongst
a group of ten horses for established genetic markers. In addition to sequencing the horse
genome, researchers generated a map of genetic variation, Single Nucleotide Polymorphisms or
SNiPs, from a variety of modern and ancestral breeds. Over 1 million Snips were mapped which
will provide valuable clues by which researchers can identify genetic contributors to physical,
behavioral and disease resistance differences. The SNiP database can be accessed at the Broad
Institute web site (http:// www.broad.mit.edu/mammals/horse.snp).

DNA Microarrays

Exploitation of information gleaned from whole genome sequencing projects, such as the human
genome project and similar programs for a large variety of animal species to date, is made
possible through the use of high density oligonucleotide microarrays, often simply called DNA
microarrays or "Gene Chips”. The technology upon which microarrays are based has been
reviewed extensively and this powerful technique has been in fairly widespread use for the past
10-12 years.2,3 Microarrays allow the measurement of the level of expression of genes in a

36

particular tissue by hybridizing labeled cellular messenger RNA (mRNA), or its corresponding
cDNA, to short oligonucleotide segments (probes) immobilized on the array surface. The mRNA
abundance of a gene (its level of expression) can then be measured by reading the signal
intensity of the label where the hybrid forms between the cellular material and the immobilized
gene.

The power of this technique lies in the fact that the expression profile of literally thousands of
genes can be measured in a single experiment, thus vastly superseding older, single gene
measurements such as Northern blot analysis and polymerase chain reaction (PCR).4 Microarray
technology has become instrumental in providing in-depth and oftentimes unexpected knowledge
of biological processes and disease pathways. Up until now, however, its use in equine
applications has been limited to smaller "custom” arrays which examine specific processes, e.g.
cartilage development.5 Furthermore, a significant number of array experiments to date have
been heterologous rather than homologous; meaning that cross-hybridizing RNA to a microarray
for a different species has been used as an index of gene expression changes in equine tissues.
Obviously, while cross-species experiments have been very useful, some transcripts can fail to
hybridize when genes are not well conserved between species and important information can be
missed as a result.

The publication and annotation of the whole equine genome will greatly add to the analytical
power of this technique as it applies to understanding and enhancing health and performance in
the horse. Much useful information, however, has been generated using microarrays in equine
applications. For example, a preliminary molecular signature of normal articular cartilage in
immature horses has been established.5 Additionally, microarrays containing human cDNAs
were used in a study to gain a better understanding of gene expression profiles in
spermatogenesis and to suggest novel therapeutic approaches to enhance stallion fertility.6
Similarly, an oligonucleotide human microarray has been used to study gene expression in
chronic obstructive pulmonary disease (COPD) (heaves)-affected horses. This study identified
several new genes which strengthened our understanding of this debilitating disease.7

Nutrigenomics

One very interesting and increasingly popular extension of microarray technology is the field of
Nutritional Genomics or "Nutrigenomics”. It has been recognized for several years that many
nutrients are not just sources of fat, vitamins, minerals, etc. but have an additional ability to act
as potent dietary signals which have the capacity to modulate gene expression in a tissue specific
fashion. Nutrigenomics attempts to determine the effects of common dietary ingredients on the
activity or expression of whole genome; thus gaining an insight into how nutritional molecules
affect metabolic pathways and homeostasis.8 The burgeoning number of publications in this area
in recent years bears testament both to the popularity of this relatively new field of investigation
and its power in dissecting the molecular mechanisms of nutrient-related health and disease
states. In recent years, the field has expanded to not only investigate the impact of classes of
nutrients on gene expression, but also the effect which different chemical forms of the same
nutrient have on transcriptional profiles in different tissues.

37

One example of this application lies in the area of nutrition and fertility; an area of prime
concern in the Thoroughbred industry. Selenium is an essential trace element which has long
been known to be a key factor in both male and female fertility. Over the years, however,
markers of selenium adequacy have been adopted which rely on blood selenium measurements
or enzyme activity assays for known seleonoenzymes, such as glutathione peroxidase. Relatively
little attention has been paid to measuring the impact of selenium on molecular markers of
fertility and no studies have examined the effects of different forms of selenium in reproductive
tissue. Nutrigenomics has opened this area to scrutiny and is generating valuable data which will
be important in a variety of species, horses included.

Dedicated microarray experiments to study the effect of selenium supplementation in horses
have not been conducted but such studies have been completed in hen oviduct; a recognized
model, cross-species tissue for the study of reproductive function.9 Our own studies10 have
revealed surprising differences in the gene expression profiles in oviduct, resulting from dietary
supplementation with inorganic sodium selenite or organic selenium, in the form of selenized
yeast (Fig. 1). For example, we have found that the organic selenium yeast source affected the
expression of key genes involved in the pathway of follicle stimulating hormone (FSH). When
compared with selenium-deficient hens, sodium selenite up-regulated the expression of one gene
in the pathway, protein kinase A (PKA) but did not elicit any change in mRNA levels of the
other genes involved in FSH action. Selenized yeast, on the other hand, up-regulated all member
genes of this critical pathway, implying more efficient FSH action. This finding may explain
why enhanced female reproductive performance is observed in several species when selenized
yeast, versus other forms of selenium, is included in the diet.

 

Figure 1. Linked effects. Follicle stimulating hormone (FSH) is a key reproductive hormone that controls
granulosa cell differentiation and development. Granulosa cells surround the developing oocyte and supply it
with steroid hormones and a host of other growth factors. They are critical, therefore, for optimal oocyte
development and maturation. Key components in the pathway of action of FSH include protein kinase A
(PKA), transcription factor SP3, and the serum glucocorticoid-regulated kinase.11

38

Conclusions

While the equine genome has just recently been elucidated and homologous arrays are not yet
available, the prospect of transcriptional analysis of equine tissues using such arrays is very
exciting indeed. From a practical perspective nutrigenomic analysis, in particular, should yield
extremely valuable information on the impact of different dietary regimens, individual nutrients
and their sub-types on key physiological processes. With this information, we can be hopeful that
specific nutrient forms and supplements will arise to enable us to improve both horse health and
performance through the simplest means possible - dietary addition.

References

1. Ramery E, Closset R, Art T, et al. Expression microarrays in equine science. Vet Immun
Immunopath 2009;127:197-202.
2. Brown PO and Botstein D. Exploring the new world of the genome with cDNA
microarrays. Nat Genet 1999;21(Suppl. 1):33-37.
3. Duggan DJ, Bittner M, Chen Y, et al. Expression profiling using cDNA microarrays. Nat
Genet 1999;21(Suppl. 1):10-14.
4. Glaser KE, Sun Q, Wells MT, et al. Development of a novel equine whole transcript
oligonucleotide GeneChip microarray and its use in gene expression profiling of normal
articular-epiphyseal cartilage. Equine Vet J 2009;41:663-670.
5. Gu W, Bertone AL. Generation and performance of an equine-specific large-scale gene
expression microarray. Am J Vet Res 2004;65(12):1664-1673.
6. Ing NH, Laughlin AM, Varner DD, et al. Gene expression in the spermatogenically
inactive "Dark” and maturing "Light” testicular tissue of the prepubertal colt. Am J
Androl 2004;25:535-544.
7. Ramery E, Closset R, Bureau F. Relevance of using human microarrays to study gene
expression in heaves-affected horses. Vet J 2008;177:216-221.
8. Mutch DM, Wahli WW, Williamson G. Nutrigenomics and Nutrigenetics: the emerging
faces of nutrition. Faseb J 2009;19:1602-1616.
9. Dougherty DC, Sanders MM. Estrogen action: revitalization of the chicken oviduct
model. Trends Endocrinol Metab 2005;16(9):414-419.
10. Power RF. 2009. Nutrigenomics: Practical applications explaining the effects of selenium
at a molecular level on hen reproductive performance. Poster presented at Alltech’s 25th
International Animal Health & Nutrition Symposium, May 17-20, Lexington, KY, USA.
11. Alliston TN, Maiyar AC, Buse P, et al. Follicle stimulating hormone-regulated
expression of serum/glucocorticoid-inducible kinase in rat ovarian granulose cells: A
functional role for the Sp I family in promoter activity. Mole Endoc 1997;11(13):1934-
1949.

39

Navigating the Sea of Supplements for Horses

Kyle Newman, PhD

Author’s address: Venture Laboratories, Inc., 2301 Maggard Drive, Lexington, KY 40511; e-
mail: knewman@ventlabs.com.

Take Home Message

A vast array of supplements is used in the equine industry. It is important to ensure that the
supplement used does not contain banned ingredients and cause disqualification of the equine
athlete. The tools to study many of these supplements now exist at the molecular level allowing
us to understand the mechanisms behind their activity. The form of many of these compounds
defines their function. Supplements have demonstrated effects on immune activity, joint health,
oxygen carrying capacity and gut health.

Introduction

As of April 5, 2010 there are 1,148 substances on the Federation Equestre Internationale (FEI)
list of prohibited substances.1 Not all of these compounds are supplements but many such as
Valerenic acid and Yohimine, have been used in both human and equine supplementation. There
are literally hundreds of supplements that are available for use in horse diets. These compounds
range from electrolytes to replenish the equine athlete to vitamins and compounds that are
designed to build muscle form and function. Because of the vast array of supplements and their
intended use, the focus here will be on products that have been shown to impact antioxidant
activity, available energy, digestive function, or joint health.

Organic Selenium

Nutrition in mineral supplementation has reached a level where mineral form dictates function.
Perhaps technologically nutritionists aren’t fully prepared for the leap from up- or down-
regulation of a gene as being a reliable predictor of animal performance, but the pieces to the
total link between gene expression and production responses are being put into place. An easy
case study is selenium supplementation. Organic selenium in the form of selenium-yeast has
been shown to improve sperm quality and integrity compared to traditional inorganic selenium
supplementation.2,3 Further, selenium yeast supplemented animals had placental expulsion in half
the time of animals receiving the same amount of inorganic selenium.4 Organic selenium
supplementation yielded higher blood, colostrums, and calf selenium than inorganic selenium.5
In horses, studies have also compared organic selenium in the form of selenium yeast to the
traditionally used sodium selenite on serum and colostrum selenium and immunoglobulin levels.4
Organic selenium supplementation improved both mare and foal serum selenium status
compared to the same levels of sodium selenite. Additionally, foal serum IgG concentrations
(measured at 12 hours) tended to be higher with organic selenium and IgG levels in foals were
significantly higher at 2 and 4 weeks and tended to be higher at every measurement. Placental
expulsion time was decreased by almost 50% in mares receiving organic selenium at 3ppm

40

compared to 3ppm selenite and nearly 100% compared to 1ppm selenite. In this study influenza
vaccination titers tended to be higher in foals from mares receiving vaccination and organic
selenium. Taking these and other selenium studies to the genomic level, preliminary studies
indicate more than 100 genes either directly or indirectly associated with reproductive processes
can be influenced by the form and level of selenium in the diet.6

Probiotics

One of the possible causes of allergies in humans is proposed to be reduced exposure to
microbial allergens as a result of our hygienic lifestyle. It is thought that probiotics may provide a
mechanism of microbial stimulation needed for developing a "normal” immune system in
infants.7 The exact mechanism of activity of probiotics on allergy is not known but differences
in the GI microflora of allergic human infants include reduced Bifidobacteria populations.8,9 The
effect of probiotics on allergies in other animal species remains to be seen. However, a strain of
Enterococcus faecium was shown to enhance immune function in supplemented dogs.10

Other investigations have demonstrated immune responses to viable micro-organisms and their
cellular components. Trials in other species have demonstrated reductions in mortality with
Bacillus subtilis supplementation.11 This may be due to the antigenic properties of the cell wall
and capsule of the Bacillus bacteria.

Prebiotics

Prebiotics are compounds that either aid in the growth of beneficial bacteria or help remove
pathogenic bacteria. Many prebiotics are complex carbohydrates. At one time, it was thought
that there were three main roles of carbohydrates in biological systems: as an energy source,
structural component (cellulose, chitin) or as glycoproteins or glycolipids and subsequently
needing to be stripped away in order to truly understand the function of the protein or lipid.
However, it turns out that the sugars on these proteins can define their function or serve to
stabilize the compound. A good example of this stabilization is industrial-grade enzyme
production where shelf-life and heat stability have been enhanced by glycosylation of the
protein. As the science of carbohydrate form and function comes more into focus, we are
learning the form of a complex carbohydrate dictates its function. Modulating the microbial
community in the gastrointestinal tract through food or feed ingredients (prebiotics) can
influence and preserve health due to the stimulation of beneficial micro-organisms but these
ingredients may also attenuate the virulence of pathogens as well as enhance the anti-adhesive
effect against pathogenic bacteria.12 The oligosaccharide receptors of the digestive cells are the
first line of defense against pathogens in the digestive tract. Pathogen receptors have strict
requirements for their ligands (proteins and glycoproteins), often consisting of a combination of
monosaccharides.12 Bacterial infection is due in many cases to the ability of bacteria to recognize
host cell surface carbohydrates and attach to these sugars. In the case of pathogens, colonization
and disease in the animal may follow. One way to prevent pathogens from causing disease is to
prevent them from attaching to the epithelial cells in the gut. Early studies using mannose in the
drinking water of broiler chicks demonstrated that this therapy could reduce the colonization rate
of Salmonella typhimurium. Purified mannose and a complex sugar called mannan
oligosaccharide (MOS) have been successfully used to prevent bacterial attachment to the host

41

animal by providing the bacteria a mannose-rich receptor that serves to occupy the binding sites
on the bacteria and prevent colonization in the animal.13,14 Mannose specific adhesins (the
binding entity on the surface of bacterial cells) are used by many gastrointestinal pathogens as a
means of attachment to the gut epithelium.

Several studies have been conducted examining the role of mannans and their derivatives on
binding of pathogens to epithelial cells in the GI tract. E. coli with mannose-specific lectins did
not attach to mammalian cells when mannose was present.15

In dogs, as well as trials in poultry, reductions in fecal clostridial concentrations have also been
noted with MOS supplementation.16,17 Although the data in the equine is lacking, the potential
for a supplement to reduce or prevent clostridial scours in the foal is attractive.

Glyco-therapy may also be part of the answer to antibiotic resistance. Perhaps some of the most
fascinating and relevant microbiology research to our industry is work on methods to reduce
antibiotic resistant bacterial concentrations using glycomic technology. Antibiotic resistance in
bacteria can be passed from one organism to another through a variety of mechanisms.18 The
increase in antibiotic resistant bacteria is partially blamed on animal production systems.19
Decreasing the prevalence of antibiotic resistant bacteria has become an important research area.
Recently, researchers at the University of Kentucky found that the prevalence of ampicillin and
streptomycin-resistant strains of Salmonella is reduced in the presence of specific yeast cell wall
preparations.18 These cell wall preparations are a rich source of mannan oligosaccharide. This
research is certainly attractive since these materials can be introduced into livestock animal diets
without toxicity or residue concerns. For the equine, using mannan oligosaccharide may remove
antibiotic resistant strains for enteric pathogens that may infect the newborn foal and thus either
prevent infection or the infective strain may be more easily treated.

Other investigators have examined complex carbohydrates as a tool against bioterrorism. A
recent trial in mice found that mice receiving yeast glucans for 1 week before anthrax infection
had doubled the survival rate of unsupplemented animals. As a therapeutic agent, mice receiving
yeast glucans had a 90% survival rate compared to 30% survival for control animals.20

Although traditionally not thought of as a role for prebiotics, studies also indicate that mannan
oligosaccharide supplementation of horses can influence immunoglobulin concentrations.
Spearman found that mares receiving MOS approximately 56 days before foaling had higher
colostral IgG and IgA than unsupplemented mares.21 Higher concentrations of colostral
immunoglobulins increases the odds for successful passive transfer to the newborn foal.

Yeast Culture and Gut Health

Impaction colic is caused by material blocking normal passage in the intestine. It can be
comprised of normal ingesta, gas, or enteroliths (stones that form around ingested twine, sand,
pebbles or other foreign material similar to pearl forming in an oyster from a foreign irritant). In
the case of impaction colic, material behind the impaction can cause distention of the intestine
which contributes to the pain the horse is feeling. Impactions can occur for a variety of reasons
including poor mastication of feedstuffs, lack of drinking water, parasite infestation, poor quality

42

hay or rapid diet changes.22,23 In some cases poor quality forage (high lignification long stem) or
very fine roughage such as coastal Bermuda grass has been implicated with colic incidence.23,24
Large colon impaction was the most frequently encountered type of chronic colic (30% of all
colic with symptoms lasting 3 days or more) observed in a British study.25 In this study, cecal
impactions were observed in less than 3% of the cases. Cohen and coworkers found colonic
impaction to represent 20.7% and 16.5% of the clinical colic cases observed by veterinarians in
Texas.26,27 Ileal and cecal impaction was diagnosed in approximately 3.3% of colic cases.26 A
follow-up to the 1995 study indicated that a change in diet (specifically hay) was associated with
colic.28 This group also implicated lower quality hay as a possible link to colonic impaction.
Later, a study confirmed these results by examining 364 horses and finding that dietary changes
such as a different batch of hay, different grain or concentrate, and decreased availability to
pasture increased the risk of colic.27

The cecum and colon of the horse is considered the major site for fiber digestion.22,29 Medina
and coworkers30 examined the effects of yeast culture on poor quality forage disappearance in
vitro when cecal and colonic fluid samples were taken from horses fed either a high fiber or high
starch diet. Regardless of whether the horses were fed the starch or fiber diet, in vitro
disappearance of the lignified fiber was greater in cultures prepared from horses receiving Yeast
culture than unsupplemented horses. These data imply that Yeast culture supplemented horses
could adapt to dietary changes more readily than those not receiving yeast culture.

The fiber degrading bacteria prefer a pH near neutrality. In fact, Stewart31 found that fiber
digestion decreases dramatically as the pH drops. The author noted a 50% decrease in cellulose
degradation when bacteria exist in an environment at pH 6.5 compared to pH 7.0. Fiber
degradation was brought to a standstill at pH below 6.0. Moore and Newman32 found that the
cecal pH was higher overall when Yeast culture was fed to ponies (6.62 vs. 6.52 for
unsupplemented ponies; P<0.05). These results were confirmed in a later study that found the
average cecal pH was greater (7.01 vs. 6.85; P<0.05), at 4, 6, and 8 hours postfeeding with Yeast
culture supplementation of a high starch diet.33 A 0.9 pH unit increase was also observed in the
colon (P>0.05). The average cecal and colonic lactic acid concentrations in the cecum and colon
of horses eating a high starch diet were also lower when the animals were supplemented with
Yeast culture. Maintenance of a higher intestinal pH may therefore increase fiber digestion
which will improve feed efficiency and maintain flow of digesta and may aid in preventing feed-
related impactions.32

Omega-3 Fatty Acids

The benefits of fish oil in human health are well documented. Many of these studies focus on
blood pressure and heart disease. There is a growing body of evidence that indicates that high
levels of docosahexanenoic acid (DHA) and eicosapentaenoic acid (EPA) provide benefits to the
exercising horse. O’Conner and coworkers observed an improvement in heart rate, serum
cholesterol and a trend toward lower lactate concentrations during exercise recovery than horses
receiving a similar amount of corn oil.34 The implications of this study are quite exciting since
time to fatigue is directly correlated with higher heart rates. The rate of use in studies of omega-
3 fatty acids in the equine focus on approximately 180 grams per day which some may argue is
high for something to be considered a supplement.

43

Joint Supplements

The work of Dorna and Guerrerro35 demonstrated that oral administration of chondroitin sulfates
yielded satisfactory results compared to the more traditional injectable route. With that came a
multitude of feed supplements on the market and many of them not meeting their label
guarantee.36 Even before these studies, evidence was present that oral administration of a
commercial joint supplement improved lameness scores.37 A study with horses with navicular
syndrome and oral administration of glucosamines also demonstrated significant improvement.38
A recent study examined undenatured type II collagen (UC-II) and found that doses of 480 mg of
a commercial product led to a reduction in overall pain and in pain with joint manipulation.39

Conclusions

Under the Dietary Supplement Health and Education Act (DSHEA), a manufacturer is
responsible for determining that the dietary supplements it produces or distributes are safe and
that any claims made are substantiated. The manufacturer is also responsible for ensuring that the
product meets the label guarantees for that compound. Many of these supplements have emerged
from the realm of witchcraft and snake oil to well documented efficacious compounds that can
aid the horse owner, improve the quality of life of the horse, and in many cases benefit the
equine practitioner. Studies at the molecular level will enhance our knowledge of these products.

References

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Dawson KA. 2006. Functional genomics: promising new tools relating nutrition to
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Spearman KR. Effect of mannan oligosaccharide (MOS) supplementation on the immune
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36.

37.

38.

39.



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