The Emerging Role of Hypoxic Training for the Equine Athlete

paper provides a comprehensive discussion on the physiological impacts of hypoxic training, its bene ts to endurance performance, and a rationale for utilizing it to improve performance in the equine athlete. All exercise-induced training adaptations are governed by genetics. Exercise prescriptions can be tailored to elicit the desired physiological adaptations. Although the applica- tion of hypoxic stimuli on its own is not ideal to promote favorable molecular responses, exercise training under hypoxic conditions provides an optimal environment for maximizing physiological adaptations to enhance endurance performance.

improve performance in the equine athlete. All exercise-induced training adaptations are governed by genetics. Exercise prescriptions can be tailored to elicit the desired physiological adaptations. Although the applica- tion of hypoxic stimuli on its own is not ideal to promote favorable molecular responses, exercise training under hypoxic conditions provides an optimal environment for maximizing physiological adaptations to enhance endurance performance. The combination of exercise training and hypoxia increases the activity of the hypoxia-inducible factor (HIF) pathway compared to training under normoxic conditions. Hypoxia-inducible factor-1 alpha (HIF-1 ) is known as a master regulator of the expression of genes since over 100 genes are responsive to HIF-1 . For instance, HIF-1-inducible genes include those critical to erythropoiesis, angiogenesis, glucose metabolism, mitochondrial bio- genesis, and glucose transport, all of which are intergral in physiological adaptations for endurance performance. Further, hypoxic training could conceivably have a role in equine rehabilitation when high-impact training is contraindicated but a quality training stimulus is desired. This is achievable through purpose-built equine motorized treadmills inside commercial hypoxic chambers. Keywords: horse; hypoxia; mitochondria; hypoxia-inducible factor; equine; normobaric; thoroughbred 1. Introduction The performance of the equine athlete consists of several variables, such as environ- mental, physiological, genetic, and training regimens. For the trainer, the major contribution to performance outcomes is the process of exercise training, particularly its structure and content. Hypoxic training involves exercising in a reduced oxygen environment. Oxygen is an important metabolite critical for maintaining a stable cellular and physiological envi- ronment and organism survival. Hypoxic training is a popular training strategy amongst human endurance athletes, as exercising or living in hypoxia improves muscle function through hypoxia-inducible factor 1 (HIF-1 )-mediated gene signaling and is linked to performance bene ts, especially in endurance-based competitions. Furthermore, temporary Animals2023,13, 2799.

Animals2023,13, 2799 2 of 11 hypoxic states may not only improve athletic performance but also provide potential health bene ts. For example, hypoxia interventions are linked to key cellular events in normal cell development and in pathological settings such as ischemia [1]. Considering that coaches and human athletes have been employing hypoxic training to gain competitive advantages for many years and that it is a topical and emerging area in racehorses, it is timely to provide a discussion on the current understanding in relation to equine athletic performance. This is necessary because often assumptions are made based on human research, and the ndings in one species may not always translate to the ndings in another. Therefore, the purpose of this commentary is to address speci c factors governing equine performance and the adaptations provided by hypoxic training that have the potential to improve performance in the equine athlete. 2. Factors Governing Performance Performance is a complex outcome involving a dynamic network of biological (genetics—nature), environmental (nurture), and biomechanical variables; here, we focus on biological variables. In humans, research has linked several genes to athletic perfor- mance, most notably insulin-like growth factor 1 (IGF1R), peroxisome proliferator-activated receptorcoactivator 1 (PPARGC1A), alpha-actinin-3 (ACTN3), and monocarboxylate transporter 1 (MCT1) [2]. In the equine eld, single nucleotide polymorphisms (SNPs) in creatine kinase (CKM) and cytochrome c oxidase (COX412) have been linked to elite Thoroughbreds; the myostatin (MSTN) gene has been shown to in uence early skeletal muscle development and aptitude for sprint racing; and the mitochondrial haplogroup L3b has been shown to have a negative association with elite Thoroughbreds [3–5]. This list is expected to expand signi cantly in the future since whole-genome sequencing technology has improved and the cost has been reduced. Today, arti cial selection of the most talented and successful racehorses, with genetic screening for optimal genetic polymorphisms, remains the current best practice for attempting to preselect performance. Conversely, envi- ronmental factors are critical to a successful athlete, none more so than exercise training. Therefore, we will limit our discussion to exercise prescription. From a physiological perspective, maximum oxygen uptake ( .

cial selection of the most talented and successful racehorses, with genetic screening for optimal genetic polymorphisms, remains the current best practice for attempting to preselect performance. Conversely, envi- ronmental factors are critical to a successful athlete, none more so than exercise training. Therefore, we will limit our discussion to exercise prescription. From a physiological perspective, maximum oxygen uptake ( . V O2max) is arguably the most important factor limiting endurance performance in both humans and horses [6,7]. . V O2maxis determined by cardiac output (stroke volume heart rate) and arterial-venous oxygen difference (a-vO2Diff). . V O2maxis primarily limited by two factors: cardiac output, or the capacity for delivering blood (oxygen) to muscles, and the capacity of muscles to utilize oxygen to transfer energy, which is dependent on mitochondrial density and function. In humans, . V O2maxhas been used as an indicator for endurance athletes' potential success. Although other physiological attributes are critical to performance in humans (e.g., lactate in ection point), a high . V O2maxis an indisputable requirement at the elite level. This, however, has not been demonstrated in horses, since the . V O2maxof Thoroughbred horses can vary from ~100 to 160 mL kg 1 min 1 [7–9]. Considering the physiological pro les of elite racehorses are not commonly reported in the literature, the performance outcomes of horses with superior . VO2maxremain to be established. In horses, ref. [7], . V O2maxcontinuously increases with training over several weeks to months, as shown in a study by Tyler et al. [7]. They examined the relationship between peripheral (skeletal muscle) and whole body ( . V O2max) adaptations to training and reported signi cant adaptations in the morphological characteristics of skeletal muscle (increases in ber area and capillarization), which were limited and largely completed by 16 weeks of training. In contrast, mitochondrial volume continued to increase throughout the 34 weeks of training and paralleled the increases in . V O2max. These ndings highlight that the markers of oxidative capacity in muscle progressively increase in parallel with aerobic capacity in response to increases in training load. This concept is supported in

limited and largely completed by 16 weeks of training. In contrast, mitochondrial volume continued to increase throughout the 34 weeks of training and paralleled the increases in . V O2max. These ndings highlight that the markers of oxidative capacity in muscle progressively increase in parallel with aerobic capacity in response to increases in training load. This concept is supported in human studies [10], which report that although oxygen delivery is the main limiting factor for . V O2max, enhanced

Animals2023,13, 2799 3 of 11 oxygen extraction fraction contributes to the remarkably high . V O2maxin endurance-trained individuals. To quantify mitochondrial oxygen consumption directly requires specialized equipment and invasive procedures, which makes it challenging to investigate the relative importance of central versus peripheral adaptation for equine performance. It is tempting to speculate that maximum mitochondrial oxygen consumption and muscle glycolytic function may limit short-distance (i.e., <5 km) at race performance, whereas lactate in ection point and running economy might be bigger predictors of endurance races (e.g., >60 km). Within skeletal muscle, the primary sites of energy production are the cytoplasm for anaerobic (glycolysis) and the mitochondria for aerobic (oxidative phosphorylation) metabolism. For the horse, however, even in high-intensity exercise, the energy is provided principally from the aerobic system and a smaller fraction from the ATP-PC glycolysis network. For example, in a horse performing at between 105 and 125% . V O2maxthe mean energy contributions are 81–72 (aerobic) and 18–27% (anaerobic), respectively [11]. During aerobic work, the energy predominantly comes from the Krebs Cycle within the mito- chondria. The mitochondria are frequently referred to as the “powerhouses” of energy production, as all aerobic energy production takes place within the mitochondria. Approxi- mately 80% of the oxygen taken up by the cells is consumed by the mitochondria to meet metabolic demands. Therefore, development of the mitochondria is a key component of training. In humans, an adaptation appears to be ber-speci c; however, most changes result predominately from endurance-based training performed below the lactate in ection point (or anaerobic threshold). It has been proposed that the magnitude of improvement in the mitochondria is in uenced by the duration of the exercise or training session [12–15]. There is, however, not a direct linear relationship, and with additional training, this factor appears to become less important. Moreover, it was also suggested that the best adaptations occur when intensity and duration of training interact. In horses, however, [7] . V O2max continuously increases with training, with signi cant increases in mitochondrial volume density also occurring in parallel with increases in . V O2,max. These ndings highlight

and with additional training, this factor appears to become less important. Moreover, it was also suggested that the best adaptations occur when intensity and duration of training interact. In horses, however, [7] . V O2max continuously increases with training, with signi cant increases in mitochondrial volume density also occurring in parallel with increases in . V O2,max. These ndings highlight that markers of the oxidative capacity of muscle progressively increase with aerobic capacity in response to increases in training load. It is well documented that the application of a stimulus or stress to the body, such as a bout of exercise, results in an acute response followed by recovery and subsequent performance improvement. In an ideal scenario, physiological adaptations would continue to occur in response to repeated training stimuli until genetics imposed a ceiling effect. The adaptation to training represents the cumulative effect of repeated bouts of exercise and is highly speci c to the exercise mode, intensity, duration, and frequency of the stimuli [16,17]. At a molecular level, adaptations to training are thought to be due to the cumulative effects of transient changes in mRNA transcripts encoding proteins that follow each acute training session [18,19]. The controlling mechanisms underlying this process are incompletely understood. The transient changes in transcription and expression of mRNA for metabolic genes have been examined following a single bout or session of exercise [20,21] and the effects on the concentration of the proteins [17,22,23]. The role of exercise-induced epigenetic modi cations in exercise adaptations is also beginning to emerge in the equine eld [24]. In addition to the training stimuli, a stimulus in the form of hypoxia may well provide additional training stress and greater physiological adaptations. 3. Hypoxic Training Is a Potent Training Stimulus The concept of altitude training became popular amongst human athletes following the 1968 Olympic Games, at which the African runners were particularly successful and were known to have trained and lived at altitude. Culminating from these observations was the concept that local tissue hypoxia is an important adaptive stress for muscle tissue in exercise training. The fundamental concept behind hypoxic

The concept of altitude training became popular amongst human athletes following the 1968 Olympic Games, at which the African runners were particularly successful and were known to have trained and lived at altitude. Culminating from these observations was the concept that local tissue hypoxia is an important adaptive stress for muscle tissue in exercise training. The fundamental concept behind hypoxic training is that the concentration of oxygen in the inspired air is reduced from 20.9% to reduced levels (e.g., 15%) with a normal atmospheric pressure (normobaric). This reduced concentration means less oxygen enters

Animals2023,13, 2799 4 of 11 the bloodstream, resulting in reduced availability for the working muscles. Living or training in a hypoxic environment ultimately unbalances the oxygen supply and demand relationship. Due to an inadequate supply of oxygen to the cells, senses detect the oxygen de ciency and activate molecular signaling pathways to correct the imbalance. The short- term response is generally physiologically based and includes changes in respiration, heart rate, blood volume, and vasodilation. Long-term changes are achieved more at the molecular level, with intermittent exposure to hypoxia primarily resulting in an up- regulation of the regulatory subunit of hypoxia-inducible factor-1 alpha (HIF-1 ) [25–27]. The HIF-1 protein is composed of two subunits, HIF-1 and HIF-1 . Generally, under normoxic conditions, HIF-1 is degraded through hydroxylation and binds to the Von Hippel Lindau (VHL) protein, resulting in proteasomal degradation of HIF-1 . However, under hypoxic conditions, this process is blocked, thus allowing HIF-1 to accumulate and bind to the subunit HIF-1 , which forms HIF-1. HIF-1 is a transcription factor that interacts with hypoxia response elements located in the nucleus of the cell, triggering transcription of target genes. HIF–1 has been referred to as a master regulator of the expression of genes, with more than 100 genes identi ed as responsive to HIF-1. For instance, HIF-1- inducible genes include those critical to erythropoiesis, metabolism, angiogenesis, glucose metabolism, mitochondria, glucose transport, and cell proliferation [26]. Importantly, the application of hypoxic stimuli alone may not elicit optimal responses for changes in gene response related to performance. The combination effect of training and hypoxia on the activity of the HIF-1 pathway has been reported to be higher than that under normoxic conditions, indicating that combining hypoxia with exercise training provides a superior stimulus. Exercise intensity provides an additional stimulus due to the increased levels of reactive oxygen species (ROS) produced during metabolic activity. The primary source of ROS production is the mitochondrial electron transport chain, which reduces the majority of oxygen to water, however, a small volume of the oxygen is converted to ROS (e.g., hydrogen peroxide or superoxide). The ROS has a

Exercise intensity provides an additional stimulus due to the increased levels of reactive oxygen species (ROS) produced during metabolic activity. The primary source of ROS production is the mitochondrial electron transport chain, which reduces the majority of oxygen to water, however, a small volume of the oxygen is converted to ROS (e.g., hydrogen peroxide or superoxide). The ROS has a role in inducing the expression of peroxisome proliferator-activated receptor-coactivator 1 (PGC-1 ), which is a master regulator of mitochondrial biogenesis, and also in in uencing increases in vascular endothelial growth factor (VEGF), a key regulator of angiogenesis [28]. Regulation of both PGC-1 and VEGF is crucial to adaptation to exercise [26,29]. However, sustained exposure to severe hypoxia has detrimental effects on skeletal muscle function, including decreases in muscle oxidative capacity and loss of muscle mass [30]. Additionally, living and training at high altitude (hypobaric hypoxia) is associated with side effects such as mountain sickness, decreased maximum heart rate and plasma volume, and impaired exercise performance in humans [31]. Conversely, short-term exposure to altitude leads to positive skeletal muscle adaptations (e.g., muscle capillary growth and mitochondrial biogenesis) [25,26,32], supporting the need to ensure that a correct balance is utilized between time spent in hypoxia and training intensity in horses. Since the early 2000s, evidence from trials has accumulated and provided insight on the in uence of various types of altitude/hypoxic training protocols, such as “living high training low” (LHTL), “living low training high” (LLTH), and “living high training high” (LHTH), on metabolic, biochemical, and other molecular adaptations. These studies were designed to establish the most ideal protocol for performance enhancement [27,33–39]. Despite some equivocal ndings on physiological and cellular responses to hypoxic train- ing among studies utilizing different training protocols, important practical applications have been suggested. Accordingly, the following sections provide a concise synthesis of outcomes from two of the most popular training methods, LHTL and LLTH, which have the most practical applications that could be adopted and guide hypoxic training in Thoroughbred horses. 4. Living High Training Low (LHTL) A seminal study by Stray-Gunderson et al. [38] examined elite male

practical applications have been suggested. Accordingly, the following sections provide a concise synthesis of outcomes from two of the most popular training methods, LHTL and LLTH, which have the most practical applications that could be adopted and guide hypoxic training in Thoroughbred horses. 4. Living High Training Low (LHTL) A seminal study by Stray-Gunderson et al. [38] examined elite male and female runners after 27 days of living at 2500 m who concurrently performed high-intensity training at

Animals2023,13, 2799 5 of 11 1250 m altitude. Despite only a 3% improvement in . V O2max, increases in erythropoietin levels and running performance were noted (8.5 0.5–16.2 1.0 IU/mL) and performance 1.1%, respectively. Several other groups reported mixed results regarding changes in . V O2maxafter training. One study [36] examined three groups of elite athletes: cross-country skiers, swimmers, and runners. Within each athlete cohort, the participants were assigned to two sub-groups: LHTL and control cohorts. Training consisted of 13–18 days at 1200 m (swimmers only had 13 days). Athletes were divided into two groups: a control group that slept at 1200 m and a hypoxic group that slept at three different altitudes: 2500 m (5 nights for swimmers and 6 for skiers and runners); 3000 m (6 nights for skiers, 8 for swimmers, and 12 for runners); and 3500 m (6 nights for skiers). Mixed results were reported for changes in erythropoiesis, with 6 days at 2500 m having little effect, whereas for 6 days at 3000 m, values were higher than basal levels. However, no signi cant increases in . V O2maxwere observed in swimmers after 13 days. Conversely, a signi cant change was reached after 18 days in runners. In another study [40], a group of male and female runners who trained for three weeks in hypoxia with train high (TH) resided at sea level, while LHTL plus train high (LH/TL + TH) stayed in normobaric hypoxia for 14 h per day. All athletes completed 4–5 h of hypoxic training per week, training at ~82% maximum heart rate. They reported that the combination of LH/TL + TH provided a greater increase in . V O2max, hemoglobin mass, and 3-km time trial performance than the TH group. Although the research highlights that the LHTL method provides some bene cial effects on performance, the logistics and associated costs make it inaccessible for many athletes [41]. Intermittent hypoxic exposure (IHE) has been proposed as an alternative form of training that involves acute 60–90 min daily exposure to hypoxia. In a study involving triathletes, participants were allocated to either a LHTL,

Discussion on hypoxic training's effects on equine athletic performance.