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Received: 23 February 2020    Accepted: 15 April 2020

DOI: 10.1111/jpn.13384

REVIEW ARTICLE

Metabolomics in equine sport and exercise

Dylan J. Klein1  | Tracy G. Anthony2,3 | Kenneth H. McKeever4

1
Department of Health and Exercise
Science, Rowan University, Glassboro, New
Jersey, USA
2
Department of Nutritional Sciences,
Rutgers, The State University of New Jersey,
New Brunswick, New Jersey, USA
3
New Jersey Institute for Food, Nutrition
and Health, Rutgers, The State University of
New Jersey, New Brunswick, New Jersey,
USA
4
Rutgers Equine Science Center,
Department of Animal Sciences, Rutgers,
The State University of New Jersey, New
Brunswick, New Jersey, USA
Correspondence
Dylan J. Klein, 201 Mullica Hill Road, James
Hall, Glassboro, NJ 08028, USA.
Email: kleind@rowan.edu
Abstract
Metabolomics is the high-throughput, multiparametric identification and clas-
sification of hundreds of low molecular weight metabolites in a biological sample.
Ultimately, metabolites are the downstream readouts of cellular signalling, transcrip-
tomic and proteomic changes that can provide a comprehensive view of tissue and
organismal phenotype. The popularity of metabolomics in human sport and exercise
has been gaining over the past decade and has provided important insights into the
energetic demands and mechanistic underpinnings of exercise and training. To the
contrary, metabolomics in the field of equine exercise physiology is lagging despite
the horse's superior aerobic and muscular capabilities, as well as its prominence in
competitive sport. As such, this narrative review aims to describe metabolomics, its
routine implementation, the various analytical methods applied and the state of its
use in the equine athlete. Sufficient attention will be paid to methodological con-
siderations, as well as gaps in the equine literature, particularly with regard to the
skeletal muscle metabolome. Finally, there will be a brief discussion of the future
directions and barriers to metabolomics use in the athletic horse. A thorough under-
standing of the metabolomics changes that occur in the equine athlete with exercise
will undoubtedly help to improve horse management and health across the lifespan.

KEYWORDS

equine, exercise, metabolomics, skeletal muscle metabolome, sport

1 | I NTRO D U C TI O N
For hundreds of years, the horse (Equus caballus) has been bred
explicitly for its superior aerobic and muscular capabilities
(Hinchcliff, 2014). As such, the horse is uniquely suited to under-
stand the effects of exercise and training on whole-body’ and
skeletal muscle metabolism. In humans, the study of muscle me-
tabolism in response to acute exercise or training has centred on a
variety of molecular mechanisms such as genomic, transcriptomic,
proteomic and signalling changes that take place following differ-
ent modes of muscle contraction (Coffey & Hawley, 2007; Egan &
Zierath, 2013). Further, measurements taken in the blood regarding
energy production and utilization (e.g. glucose, lactate and free fatty
acids) have also become a staple of metabolic exercise physiology
investigations (Brooks & Donovan, 1983; Henriksson, 1991; O'Neill,
Watt, Heigenhauser, & Spriet, 2004). Together, these insights have
provided salient mechanistic information regarding whole-body’
exercise adaptations and the plasticity of skeletal muscle that have
since been translated into nutrition and training interventions aimed
at maximizing health and performance across the lifespan (Egan &
Zierath, 2013; Hawley, Hargreaves, Joyner, & Zierath, 2014).
Most studies examining the metabolic effects of exercise in
horses have primarily focused on blood-related measures (e.g. lactate,
amino acids and free fatty acids; Greenhaff, Harris, Snow, Sewell, &
Dunnett, 1991; Poso, Essen-Gustavsson, Lindholm, & Persson, 1991;
Snow & Mackenzie, 1977b), as well as muscle fibre-type populations
and metabolic/oxidative enzymes that produce force and chemical en-
ergy needed for locomotion (Rivero & Piercy, 2014). A limited number
of targeted compounds that are involved in intermediary metabolism
and energy production and utilization have been routinely studied

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KLEIN et al.
in equine muscle as well (Harris, Marlin, Snow, & Harkness, 1991;
McGowan, Golland, Evans, Hodgson, & Rose, 2002; Snow, Harris, &
Gash, 1985; Snow & Mackenzie, 1977a, 1977b). Studies using more
sophisticated methods such as stable isotopic tracers and transcrip-
tomic analysis have recently been incorporated into equine nutrition
and exercise physiology studies. These reports have shown alter-
ations in muscle anabolic signalling and whole-body’ protein synthe-
sis/ turnover across the lifespan (Wagner & Urschel, 2012), as well
as global gene expression patterns in muscle following fatiguing ex-
ercise and training (McGivney et al., 2010; Ropka-Molik, Stefaniuk-
Szmukier, Z˙ukowski, Piórkowska, & Bugno-Poniewierska, 2017; te Pas
et al., 2013). Proteomic analysis in equine skeletal muscle also supports
transcriptomic observations and shows an increase in biopsy proteins
related to oxidative metabolism, free fatty acid uptake and utiliza-
tion, and increased energy storage after chronic exercise (Bouwman
et al., 2010). Because metabolites are downstream of cellular signalling,
transcriptomic and proteomic events, comprehensive metabolite cat-
aloging through “-omics”-based technologies can provide even greater
insights into the mechanistic underpinnings of exercise and its effects
on performance and athletic phenotype.
While metabolomics research has been conducted in humans in
a variety of sport and exercise settings for over a decade (Heaney,
Deighton, & Suzuki, 2017), the literature relating metabolomics to the
equine athlete is fledging (approximately 5 years). Nevertheless, its
utility is becoming increasingly acknowledged (Le Moyec et al., 2014,
2019; Luck et al., 2015; Mach et al., 2017). As it stands, the horse rep-
resents the least studied of the livestock animals (i.e. bovine, porcine,
ovine, caprine, equine) with regard to metabolomics-based analyses
(Goldansaz et al., 2017) despite its large muscle mass and prominence
in competitive sport. As such, this narrative review aims to describe
metabolomics and its promise in equine sport and exercise. Obviously,
the authors recognize that metabolomics can afford other opportuni-
ties related to the horse, such as animal health, disease management
and animal reproduction. In this regard, interested readers are directed
towards a recent review for such topics (Goldansaz et al., 2017). Within
the present review, a brief discussion of metabolomics platforms and
analytical methods is presented, followed by the current state of me-
tabolomics analysis in the athletic horse. References will also be made
to the relevant comparative human literature when necessary. Finally,
we will discuss the current gaps in the equine exercise metabolomics
literature, particularly with regard to the skeletal muscle metabolome,
as well as the future implications and barriers of metabolomics in
equine sport and exercise. Our ultimate goal is to highlight the promise
that metabolomics can provide to researchers, veterinarians and the
racing industry with the hopes that metabolomics becomes increas-
ingly feasible and utilized with regard to the equine athlete.
2 |  M E TA B O LO M I C S : A N OV E RV I E W
metabolomics is the large-scale, multiparametric study of low mo-
lecular weight metabolites (<1 kDa; e.g. amino acids, nucleotides and
lipids) within a biological sample (e.g. blood or skeletal muscle) using
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      141
advanced analytical chemistry techniques. Through the broad charac-
terization of these metabolites (i.e. the “metabolome”), researchers can
gain a comprehensive insight into tissue and organismal phenotype and
their interactions with a given stimuli that would otherwise take count-
less individual assays for metabolite identification and quantification.
Like other sophisticated –omics methods of determining gene or
protein expression patterns, metabolomics can be targeted (biased)
or untargeted (unbiased). Based on the assay of choice, a relatively
limited number (approximately 50) of pre-determined metabolites
can be identified and quantified (targeted), or every identifiable me-
tabolite in a sample (potentially > 500) can be semi-quantified and
inventoried (untargeted). Both approaches have their advantages
and limitations. For example, an untargeted approach can unbiasedly
screen for all metabolites in a sample and the resultant “signature”
can then be used to identify novel biomarkers that are associated
with physiological state such as training status (Duft et al., 2017), or
with other performance-related variables such as maximal aerobic
capacity (Lustgarten et al., 2013).
Conversely, a targeted approach typically screens for a limited
number of pre-defined analytes that can be quantified in absolute
concentrations or their rate of flux can be measured within a biolog-
ical system. As such, the success of the study is highly dependent
upon pre-existing knowledge about the organism–stimuli interac-
tion and the strength of the hypothesis that is being tested (Jang,
Chen, & Rabinowitz, 2018; Liu & Locasale, 2017). In either event,
metabolomics can be a highly effective and comprehensive way of
interrogating metabolic networks that are downstream of cellular
signalling, genomic, transcriptional and proteomic events.
The amount (i.e. mg or ml) and type of tissue are also important
considerations with regard to metabolomics analyses (Foroutan,
Goldansaz, Lipfert, & Wishart, 2019). While the amount of tissue
needed for metabolite identification has decreased over the years (now
even single cells can be analysed), sample type (tissue or biofluids) and
the metabolites of interest (e.g. polar metabolites or non-polar lipids)
will ultimately dictate how the sample is prepared for instrumentational
analysis (Jang et al., 2018). For an in-depth review on sample prepara-
tion concerns, the reader is referred the following papers (Foroutan
et al., 2019; Jang et al., 2018; Liu & Locasale, 2017). The ultimate goal
of sample collection is to obtain a sufficient amount of tissue while also
preserving the physiological state of the tissue as best as possible. This
typically means collecting ~100 µl of biofluid and ~100 mg of tissue.
Following collection, rapid cooling of biofluids on ice or “snap freezing”
tissues/biopsies in liquid nitrogen is recommended in order to quench
enzymatic and chemical activities that can affect metabolite purity and
integrity. Further, practicing aseptic techniques will also ensure that
samples are not polluted with foreign contaminants or xenobiotics that
can also be identified via metabolomics platforms.
3 | M E TA B O LO M I C S PL ATFO R M S
Currently, there are two commonly used platforms for metabo-
lomics analysis: nuclear magnetic resonance (NMR) spectroscopy
 |
142       KLEIN et al.
and mass spectrometry (MS). Typically, MS-based analysis is cou-
pled to either liquid or gas chromatographic techniques (LC and
GC, respectively) for molecule separation prior to metabolite
identification. Ideally, both LC and GC are employed to increase
throughput and obtain a greater number of metabolites. NMR and
MS methodologies each have their strengths and limitations, and
a brief discussion is provided below with additional information
provided in Table 1.
3.1 | Nuclear magnetic resonance (NMR)
spectroscopy
NMR spectroscopy utilizes a high-powered magnet to measure the
intrinsic spin of atomic nuclei that can ultimately elucidate a mol-
ecule's structure and identity. The power in NMR spectroscopy as
a metabolomics platform is that it can also quantitate the number
of nuclei in a compound, and therefore its absolute concentration.
The major limitation with NMR is that not all isotopes exhibit nuclear
spin and thus cannot be identified via this platform. As such, NMR is
limited in characterizing metabolomics signatures on a global tissue
scale.
3.2 | Mass spectrometry (MS)
Mass spectrometry identifies compounds based on their molec-
ular weight after first derivatizing (e.g. ionizing) the compounds
within the sample and separating them based on their charge, as
well as their retention times through a column (interpreted as a
mass-to-charge ratio [m/z]). Unlike NMR, MS is highly sensitive,
has a higher throughput and can identify hundreds of individual
compounds on a global scale. As aforementioned, MS is typically
coupled to gas and/or liquid chromatographic methods thereby
increasing the separation of a variety of metabolites within a sam-
ple that can be sensitively and accurately identified. Ultimately,
once spectral or chromatograph peaks are generated, metabo-
lites can then be identified and/or quantified for statistical and
TA B L E 1   metabolomics platforms and their advantages and limitations
Platform Advantages
NMR • Preserves metabolite integrity
• Provides greater structural information
• Sample can be reanalysed
• Absolute quantitation possible
• No need for gas or vacuum supply
MS (coupled • High sensitivity
to GC or LC) • Overlapping metabolite peaks can be discriminated
via m/z
• Broad metabolite coverage
• Analysis of gas or volatile compounds (GC)
• Superior compound separation (LC)
Abbreviations: GC, gas chromatography; LC, liquid chromatography; m/z, mass-to-charge ratio; MS, mass spectrometry; NMR, nuclear magnetic
resonance spectroscopy.
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bioinformatic analyses. For an in-depth review of data preprocess-
ing, readers are encouraged to consult the following (Dyar, Artati,
Cecil, & Adamski, 2019).
4 | B I O I N FO R M ATI C A PPROAC H E S
Due to the high-throughput and multiparametric nature of metabo-
lomics studies, it is required to have high-powered computational
tools in order to synthesize, analyse and visualize the date. Numerous
bioinformatic approaches can be used for metabolomics-based
analysis that range from simply identifying meaningful metabo-
lites to placing groups of metabolites into a pathway or physiologi-
cal context. Herein, we will briefly describe a handful of commonly
used multivariate approaches that can compare the metabolomics
profiles of pre-defined states (e.g. pre-exercise vs. post-exercise or
trained vs. untrained). These approaches are principal component
analysis (PCA), orthogonal partial least squares-discriminant analysis
(OPLS-DA), hierarchical clustering and pathway enrichment analysis.
As with every approach in metabolomics, the strength of the analy-
sis will be a direct function of hypothesis being tested. Thus, cer-
tain bioinformatic approaches may be more or less prudent, and this
should be under the discretion of the investigator.
4.1 | PCA and OPLS-DA
Both PCA and OPLS-DA are powerful modelling tools that help to
visualize, either two or three dimensionally, the totality of a metab-
olomics data set with regard to the biological or physiological state
of the samples. This ultimately allows for a high-level view of the
distribution of the data set, which can provide meaningful informa-
tion with regard to discriminating how individual samples (e.g. phys-
iological states) differ from one another (Worley & Powers, 2013).
Such interpretations could involve the metabolomics profile be-
tween pre-race and post-race in Standardbred harnessed racing, or
between metabolomics signatures that distinguish elite from non-
elite horses.
Limitations
• Lower sensitivity
• Complex sample profiles may overlap; cannot discriminate
• Expensive to purchase; specialized training required
• Lower metabolite coverage
• Destroys sample integrity; cannot be reanalysed
• Subject to instrument fluctuations
• Gas and vacuum supply needed
• Expensive; specialized training needed
• Absolute quantitation requires internal standards
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KLEIN et al.       143

4.2 | Hierarchical clustering 5 | E X E RC I S E M E TA B O LO M I C S : FRO M

Hierarchical clustering is an unsupervised method for organizing the
data and can show large-scale differences in the data set (Worley,
Halouska, & Powers, 2013). There are several types of hierarchi-
cal clustering approaches, and many “distance metrics” that can be
relied upon. A common method is complete clustering using the
Euclidean distance, where each sample is a vector with all of the me-
tabolite values. In the end, each cluster that is produced is distinct
from another, but the samples within each cluster are more similar to
each other. As such, inferences can be drawn about the similarities
and differences between physiological states surrounding exercise
or competition.
4.3 | Pathway enrichment analysis
Pathway enrichment analysis is common in –omics research (Garcia-
Campos, Espinal-Enriquez, & Hernandez-Lemus, 2015) and can allow
for the identification of compounds that are overrepresented within a
pathway as a function of the entire metabolomics data set. There are
a number of freely available, open-source softwares (e.g. Cytoscape
or MetaboAnalyst) that allow for data visualization and pathway en-
richment. As such, certain metabolites and pathways that encom-
pass a host of altered metabolites can be highlighted and may offer
unique insights into phenotype. One limitation is that the enrichment
analysis treats each metabolite within a metabolic pathway with equal
weight/importance, regardless of potential metabolite–metabolite or
metabolite–protein interactions. Further, it assumes that metabolic
pathways operate in isolation from each other. This notion is counter
to what is understood about metabolism and how pathways overlap.
Therefore, caution must be taken when interpreting overrepresenta-
tion results, and sufficient pre-existing biological knowledge about a
system must be a factor when analysing pathway enrichment data.
H U M A N TO H O R S E
Since 2007, targeted and non-targeted NMR and MS-based metabo-
lomics research has been increasingly conducted in humans in a vari-
ety of sport and exercise-related settings (Heaney et al., 2017). These
observations have largely centred on changes in the abundances
of metabolites in blood, urine and saliva in response to acute exer-
cise or training (Heaney et al., 2017; Nieman et al., 2014; Pohjanen
et al., 2007). Such investigations have afforded the opportunity to
identify novel biomarkers associated with exercise that have helped to
better understand the underlying mechanisms that promote recovery
and the beneficial adaptations that occur with training. Therein, stud-
ies in humans have shown novel changes in metabolite classes related
to amino acid, lipid, carbohydrate and energy metabolism following a
variety of exercise intensities and modalities (e.g. endurance exercise
and resistance exercise; Berton et al., 2017; Nieman, Shanely, Gillitt,
Pappan, & Lila, 2013; Valerio et al., 2018). Subject populations have
also spanned a wide range of fitness levels, from sedentary/untrained
individuals (Huffman et al., 2014; Lewis et al., 2010) to highly trained,
elite athletes (Al-Khelaifi et al., 2018; Yan et al., 2009). The vast ma-
jority of research has centred on the effects of exercise or training
on changes in blood, urine and/or saliva metabolomics signatures
(Heaney et al., 2017), likely due to the non-invasiveness of obtaining
these biofluids. While blood, saliva and urine measures may be easy to
obtain, there is evidence that blood and muscle metabolomes share
very little overlap (Fazelzadeh et al., 2016), thus questioning the util-
ity in metabolomics biofluid measures to understand exercise-induced
muscle alterations. As such, care must be taken when interpreting the
results of blood-derived data that encompass metabolite uptake and
release from a host of organ tissues.
Currently, there is a dearth of exercise-related metabolomics
studies in the athletic horse (Table 2). The majority of reports have
been limited to changes in plasma metabolomics parameters in

TA B L E 2   Summary of exercise-related metabolomics studies in the athletic horse

Authors Platform Tissue(s) Breed Sample size Exercise

Le Moyec et al. (2014) NMR Plasma AR n = 28 Endurance race (130–160 km)

Luck et al. (2015) NMR Plasma AR n = 46 (young); n = 11 (mature) Endurance race (90–160 km)
Jang et al. (2017) NMR Plasma, muscle, TB n = 3 30-min bout of exercise (?)
urine
Mach et al. (2017) NMR Plasma AR n = 10 Endurance race (160 km)
Le Moyec et al. (2019) NMR Plasma AR n = 16 (90 km); n = 15 (120 km); Endurance race (90–160 km)
n = 9 (160 km)
Ueda et al. (2019) NMR Plasma TB n = 60 a  Jockeyed race (times/distances
not reported)
Klein et al. (2020) UHPLC-MS Muscle SB n = 8 Incremental exercise test;
12 weeks training (aerobic/
anaerobic)

Abbreviations: AR, Arabian; NMR, nuclear magnetic resonance spectroscopy; SB, Standardbred; TB, Thoroughbred; UHPLC-MS, ultra-high-
performance liquid chromatography-mass spectroscopy.
a
Number of total samples (i.e. from 30 horses pre-race, and 30 horses post-race).
 |
144       KLEIN et al.
response to endurance riding events (Le Moyec et al., 2014, 2019;
Luck et al., 2015; Mach et al., 2017), with urine and muscle tissues
being far less described (Jang et al., 2017). Indeed, to the best of our
knowledge, only two published metabolomics studies have been con-
ducted in equine skeletal muscle (Jang et al., 2017; Klein, McKeever,
Mirek, & Anthony, 2020), and only one having investigated the effects
of longitudinal training on the equine metabolome (Klein et al., 2020).
In the first equine metabolomics study, using an NMR-based ap-
proach, Le Moyec and company (Le Moyec et al., 2014) showed in
Arabian and half-breed Arabians that long endurance racing (races
of 120–160 km) strongly affects plasma lipid and amino acid me-
tabolite signatures. In addition, it was observed that pre-race and
post-race profiles could not effectively discriminate against horses
that finished and those that were disqualified. While these findings
potentially suggest a limited role in blood-derived metabolites for
predicting endurance race outcomes, it should be noted that the
study was not designed for discriminating finishers from non-fin-
ishers. Nonetheless, of the horses that finished the race and were
ranked, lipid biomarkers correlated highly with average racing speed,
showing some promise in biomarker discovery for performance-re-
lated outcomes in elite endurance racehorses.
In a follow-up analysis to their 2014 paper (Le Moyec et al., 2014),
Luck et al. (2015) extend upon their previous findings and compared
the plasma metabolomics profiles between young (<6 years) and
experienced (>8 years) Arabian and half-breed Arabian endurance
racing horses and observed distinct differences in glucose and lipid
metabolism following 90 and 160 km endurance rides. Interestingly,
more experienced horses were able to maintain post-race glycaemia
with higher levels of lactate and creatine, and lower levels of lipid-re-
lated metabolites and glutamate following 160 km than less expe-
rienced horses following a 90-km race (Luck et al., 2015). As such,
this study underscores the utility of metabolomics in highlighting the
differences in metabolism between younger, naïve racehorses and
those that are slightly older and have greater racing experience.
In a second follow-up to their 2014 study (Le Moyec et al., 2014),
the same group reanalysed the blood metabolomes of endurance
horses alongside alterations in blood transcriptomes and miRNomes
(Mach et al., 2017). The authors documented distinct changes in 11
metabolites, 263 genes and 5 miRNAs that are related to glucose ho-
meostasis, lipid metabolism, ketone body generation, ATP synthesis
and increased acetate production. Moreover, using a systems biol-
ogy approach, they were able to discriminate horses that finished
the 160-km race from disqualified horses. This revealed greater
metabolic and inflammatory issues pre-race in the non-finishers that
likely made it more difficult for these horses to handle the increased
physiological demands of energy production and inflammatory me-
diation during the event. Taken together, this study revealed the
superiority of a systems biology approach in effectively predicting
racing performance by integrating metabolomics and transcriptomic
(mRNA and miRNA) profiles for diagnostic biomarker discovery.
In their most recent study, Le Moyec et al. (2019) investigated
the impact of racing distance (90 km vs. 120 km vs. 160 km rides)
on plasma metabolomics signatures, mainly in Arab and Half Arab
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endurance horses. Through their NMR analysis, it was shown that
racing distance had a significant effect on plasma metabolomics sig-
natures with the longest distance (i.e. 160 km) having the greatest
impact on carbohydrate, lipid and protein metabolic markers. These
signatures indicated a shift from carbohydrate to lipid metabolism to
support glycaemia, as well as a greater propensity for longer dura-
tion racing to promote protein catabolism and degradation.
Given the aforementioned studies, it is clear that the physiological
and energetic demands of endurance racing have an understandably
robust effect on the equine plasma metabolome. This has been shown
through several investigations showing alterations in metabolites re-
lated to protein and lipid metabolism that are likely elevated in order
to maintain glycaemia during endurance races. Additional factors such
as training status and racing distance are likely to also play pivotal
roles in the metabolomics profiles identified, and future independent
studies are strongly needed that incorporate measures of tissues
other than blood during these as well as other types of racing events.
In one of the few studies that evaluated the effects of acute exer-
cise on the equine skeletal muscle metabolome (in addition to blood
and urine; Jang et al., 2017), the authors identified 35 metabolites
using NMR spectroscopy and showed exercise-induced alterations
related to amino acid and energy metabolism. Unfortunately, serious
methodological limitations exist such as the paltry sample size (i.e.
n = 3) and the failure of the authors to describe the 30-min exercise
bout (i.e. modality or intensity). Taken together, it is difficult to ascer-
tain any meaningful information from this trial. More recently, in the
only non-targeted metabolomics study in the horse, our group re-
cently published the effects of acute fatiguing exercise and training
on the skeletal muscle metabolome of Standardbred horses (Klein
et al., 2020). Our results indicated that amino acid, lipid, nucleotide
and xenobiotic-related metabolites play pivotal roles in the skeletal
muscle response to acute fatiguing exercise and training. Because it
has been observed in humans that blood and muscle metabolomes
show very little overlap (Fazelzadeh et al., 2016), more research is
needed with respect to the equine muscle metabolome and how it
relates to blood measures during exercise and sport.
With regard to the horseracing industry, metabolomics analysis
of blood plasma was recently employed to effectively discriminate
samples of horses before and after a jockeyed race at Japanese racing
testing centres (Ueda, Tozaki, Nozawa, Kinoshita, & Gawahara, 2019).
Because most drug tests for illicit doping take place peri-race and op-
erate to find pre-determined compounds of known molecular size and
weight (Peters et al., 2010), non-targeted metabolomics analysis can
be employed to indiscriminately find compounds in designer drugs
that may be unknown or underappreciated. More research is strongly
warranted in this area given the risk of drug abuse in equine sport.
6 | A D D ITI O N A L G A P S I N TH E EQ U I N E
LITE R AT U R E
Given its infancy, equine exercise metabolomics leaves much to be
desired. The present section is meant to briefly summarize some

KLEIN et al.
of the most glaring limitations related to equine metabolomics.
For a more comprehensive overview of livestock metabolomics
limitations, the reader is referred to the following (Goldansaz
et al., 2017).
6.1 | Exercise and sport modality
First, more studies are needed regarding different types of equine
sport, exercise and competitions (e.g. jockeyed racing, harnessed
racing, jumping and eventing, and polo matches). Given the unique
locomotive and energetic demands of each sport/event, it is logi-
cal to hypothesize that global metabolism will be differentially al-
tered therein. Moreover, the unique training paradigms employed
for different types of equine competition for muscle conditioning
(Rivero, 2007) could also likely alter blood and muscle metabo-
lomics signatures that are distinct to that competitive pursuit. As
such, unique metabolomics signatures that are reflective of various
competitions and training paradigms could lend crucial informa-
tion into nutritional strategies that can promote performance and
recovery.
6.2 | Training status and overtraining signatures
Currently, there are only a handful of investigations that have ex-
amined the effects of racing experience and/or training status on
the equine metabolome (Klein et al., 2020; Luck et al., 2015), and no
studies have been conducted examining overtraining or overreach-
ing on the equine metabolome, be it in blood, urine or skeletal mus-
cle. Identifying a biofluid or muscle-specific training signature could
provide useful information regarding whole-body’ or tissue-specific
metabolism following periods of training or detraining, or between
elite and non-elite racehorses. Further, metabolomics signatures
could provide useful information regarding overtraining that can aid
in better horse management during intensive training periods.
6.3 | Breed differences
Breed differences are also likely to impact metabolomics signatures,
as each breed has been selected for its intrinsic athletic prowess,
typically explained as a function of muscle fibre-type and fibre dis-
tribution patterns (Rivero & Hill, 2016). Thoroughbreds and Quarter
Horses represent elite sprinters, whereas Standardbreds and
Arabians represent superior middle and long-distance runners, re-
spectively. While these are four of the most studied of the athletic
breeds, Arabians and endurance horses have garnered a large share
of the metabolomics literature given their innate ability to run for
long distances (e.g. 160 km races) and the enormous energetic de-
mand that endurance exercise places on whole-body’ metabolism.
More research is obviously needed in this area to delineate the im-
pacts of breed on metabolomics profiles.
|
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      145
6.4 | Methodological considerations
Two methodological considerations paramount to metabolomics
study are sample size and the time point of sample collection sur-
rounding exercise, a race or competition. Indeed, most equine
metabolomics investigations have been conducted in sample sizes
≤30 animals (Table 2). Increasing the feasibility of sample collection
and analysis will certainly allow for greater subject sample sizes.
Additionally, depending on the time point(s) chosen, metabolomics
signatures may reflect the energetic demands of the bout of exercise
itself or the metabolic processes of the early (1–3 hr) or late (24–
48 hr) post-exercise periods that are indicative whole-body/tissue
recovery and repair. As such, the metabolites profiled may be more
or less a readout of immediate tissue signalling (short term) and/or
indicators of longer-term transcriptomic and proteomic changes.
6.5 | Platform considerations
Finally, analytical instrumentation also presents a current limitation
regarding equine metabolomics studies as a whole. To date, NMR
analysis has been employed most often, with our group publish-
ing the first metabolomics report using an untargeted, UHPLC-MS
approach (Table 2). While NMR spectroscopy can be a powerful
method for metabolite quantification and detection, a MS platform
coupled to GC and/or LC methods is more sensitive and can identify
a greater number of metabolites with a higher degree of sensitivity
(as discussed above). As such, more studies are needed regarding
MS-based metabolomics analysis and global metabolomics profiles.
7 | FU T U R E D I R EC TI O N S , BA R R I E R S A N D
I M PLI C ATI O N S
Since its inception, metabolomics has come leaps and bounds with
regard to workflow feasibility and metabolite identification tech-
niques (Monteiro, Carvalho, Bastos, & Guedes de Pinho, 2013).
The field of equine science is currently behind with regard to me-
tabolomics analysis, and strong efforts should be made to increase
its utilization in equine sport and exercise, particularly with regard
to the skeletal muscle metabolome. As pointed out in a recent
review (Goldansaz et al., 2017), many agricultural livestock insti-
tutions may not be equipped with metabolomics technologies/
facilities (i.e. NMR spectroscopy or MS platforms) thereby hinder-
ing its widespread use. Further, while the overall metabolomics
fingerprint of a tissue or fluid is valuable in its own right, it must be
acknowledged that metabolites are ultimately functions of path-
ways that change in their fluxes and rate of metabolite production
and utilization (Jang et al., 2018). To gain a truly comprehensive
view of organismal or tissue phenotype, a systems biology ap-
proach that couples metabolomics to other sophisticated –omics
technologies (i.e. transcriptomics, miRNomics, proteomics) and
isotopic tracer methodologies that can interrogate tissue fluxes
 |
146       KLEIN et al.
will be most salient. Indeed, one study in horses has already shown
the promise of a systems biology approach for predicting racing
performance based on metabolomics, transcriptomic and miR-
Nomics analyses (Mach et al., 2017).
Another huge limitation of metabolomics analysis is the inabil-
ity to delineate the tissue-specific contribution or subcellular com-
partmentalization of metabolites and their respective pathways (e.g.
muscle vs. liver's contribution to the blood metabolome or cytosolic
vs. mitochondrial metabolism within a cell). Indeed, the inherent na-
ture of metabolomics and its “snap shot” of metabolism provide the
sum of metabolites within a fluid or a number of intracellular organ-
elles, which might give a misleading view of whole-body’ or tissue
phenotype. While isolating individual organelles (e.g. mitochondria
vs. endoplasmic reticulum) and profiling each compartment might
seem ideal, in practice it would be nearly impossible to achieve such
separation without compromising the physiological and metabolic
state of these structures. Novel adaptations of tracer methodologies
may be able to resolve some of these issues, as has been shown by
using hydrogen tracing and characterizing NADPH-dependent me-
tabolism within the cytosol versus mitochondria (Lewis et al., 2014).
Further, flux analysis can be used to interrogate the contribution
of certain tissues to whole-body’ metabolism (Engelen, Ten Have,
Thaden, & Deutz, 2019), thus providing much needed context to
blood-derived measures.
8 |  CO N C LU S I O N S
The athletic horse, given its large muscle mass and athletic prowess,
uniquely positions itself as model organism to understand exercise
metabolism. metabolomics-based analyses of biofluids and tissues
can build upon what is already known about this elite competitor
and serve to improve nutrition, training, and health and performance
across the lifespan. Currently, a handful of metabolomics studies
have begun to elucidate the impacts of exercise on equine metabo-
lism and performance, but much is left to uncover. To improve our
scientific understanding of the horse's response to exercise and
training, future studies should aim to utilize platforms outside of
NMR for greater metabolite detection, as well as the coupling of
metabolomics with additional –omics methodologies, particularly in
skeletal muscle.
C O N FL I C T O F I N T E R E S T
The authors declare no conflicts of interest.
AU T H O R C O N T R I B U T I O N S
D.J.K. wrote the paper and had primary responsibility for final con-
tent; D.J.K., T.G.A. and K.H.M. edited and revised the manuscript. All
authors read and approved the final manuscript.
A N I M A L W E L FA R E S TAT E
The authors confirm that the ethical policies of the journal, as noted
on the journal's author guidelines page, have been adhered to. No
14390396, 2021, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jpn.13384 by CAPES, Wiley Online Library on [06/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ethical approval was required as this is a review article with no origi-
nal research data.
ORCID
Dylan J. Klein  https://orcid.org/0000-0002-3936-1861
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How to cite this article: Klein DJ, Anthony TG, McKeever KH.
Metabolomics in equine sport and exercise. J Anim Physiol Anim
Nutr. 2021;105:140–148. https://doi.org/10.1111/jpn.13384