ADAPTATIONS OF RACING THOROUGHBREDS TO A HIGH-ALTITUDE CHAMBER: A PILOT STUDY By Jie Li A THESIS Submitted to 2020 Michigan State University in partial fulfillment of the requirements for the degree of Animal Science—Master of Science ADAPTATIONS OF RACING THOROUGHBREDS TO A HIGH-ALTITUDE CHAMBER: A PILOT STUDY ABSTRACT Jie Li By Recently, considerable attention has been paid to “living high and training low” due to athletic performance being enhanced with acclimation to low oxygen conditions while training in the presence of normal oxygen concentrations. While a number of physiological adaptations to high altitude in humans have been published, relatively few reports exist regarding the responses of horses to similar conditions. The goal of this work was to evaluate whether horses could be acclimated to a high-altitude chamber (HAC) and to monitor the performance of those horses. ACKNOWLEDGMENTS First and foremost, I would like to thank the members of my committee: Drs. Brian Nielsen, Harold Schott, Holly Spooner, and Lawrence Olson. Thanks for your guidance and support for the last two years. I cannot thank you enough for your contributions to my project and my education here at MSU. I sincerely thank Dr. Brian Nielsen for his guidance and encouragement in carrying out this project work over the last two years. It has been a lovely experience working with you. I clearly remember when I get the official offer letter and how excited I was. I appreciate that you accept me to your team. I know I am not good at English writing. And try to meet every deadline. Honestly, I feel stressed when seeing other team members did excellent jobs. I would never have believed I was worthy of working with such an excellent horse rider and a remarkable educator. However, your constant optimism lights me up in the darkest of times. I faced many personal and academic challenges in my time here at MSU, but your high expectations make me move forward. I have no words to express my appreciation for your help you give to me during my school year at MSU. I also would like to thank other members form Spartan Equine Research Team members, Dr. Cara Robison always gives me constructive suggestion about my project work and data analysis. I would never have made it through without you; Abby, Alyssa, and Fernando are lovely office mate also help me with my class and study. Finally, to my family and friends, I appreciate my parents provide financial support for me and continuously believe, console, and encourage me to jump to my iii comfort zone. Due to the different time zone, you are always there to care about my life no matter how busy you are. No words can express my appreciation to you. I love you so much!! iv TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii INTRODUCTION .............................................................................................................. 1 CHAPTER 1. Literature Review ........................................................................................ 3 Equine physiological and metabolic responses to exercise ......................................... 3 Blood doping ................................................................................................................ 4 Blood doping scandals in sports .................................................................................. 4 Blood transfusion ......................................................................................................... 5 Blood transfusion’s physiological effects .................................................................... 6 EPO’s role in erythropoiesis ........................................................................................ 6 Doping with EPO ......................................................................................................... 7 Cobalt ........................................................................................................................... 8 Cobalt’s role in blood doping ....................................................................................... 9 Cobalt’s threshold concentration and elimination........................................................ 9 Cobalt’s role in erythropoiesis ................................................................................... 10 Blood doping detections .............................................................................................11 Altitude training ......................................................................................................... 12 Elevations in training ................................................................................................. 13 Physiological acclimations in human and laboratory animals ................................... 13 Can horses adapt to hypoxic environment? ............................................................... 14 Duration of hematological acclimations .................................................................... 15 Previous studies in horses .......................................................................................... 16 High-altitude chamber ............................................................................................... 16 CHAPTER 2. Effects of “High Altitude Chamber” on Racing Thoroughbreds ............... 18 Materials and methods ............................................................................................... 18 Statistics ..................................................................................................................... 22 Results ........................................................................................................................ 22 CHAPTER 3. Discussion and Conclusion ........................................................................ 27 Discussion: ................................................................................................................. 27 Conclusion: ................................................................................................................ 32 REFERENCES ................................................................................................................. 33 v LIST OF TABLES Table 1. Serum iron and TIBC in the first two Thoroughbreds after 4-wk supplementation before the study…………………………………………………………………………. 22 Table 2. Resting Hb (g/dL) of two Thoroughbreds in trial 1…………………………… 23 Table 3. Hemoglobin concentration (Hb) in two Thoroughbreds after SET on day 0, 10, and 31…………………………………………………………………………………… 23 Table 4. Average HR in two Thoroughbreds after SET on day 0, 10, and 31…………... 24 Table 5. The average finish position and average speed rating score both prior to HAC acclimation and after HAC acclimation in four Thoroughbred racehorses along with the number of races from which the average was determined……………………………… 26 vi LIST OF FIGURES Figure 1. Means of Hb on day 0, 10, and 31. Means not sharing similar superscripts differ (P < 0.05)………………………………………………………………………………... 24 vii INTRODUCTION Since the 1968 Olympic Games in Mexico City (elevation 2,240 meters), when the benefits of altitude training on athletic performance became evident (Kasperowski, 2009), people started to use hypoxic conditions to improve aerobic performance in endurance training. It has been recognized that boosting the oxygen transport capacity, either by training or through other means commonly referred to as “blood doping,” is the most powerful tool for improving athletic performance in aerobic sports (Segura and Lundby, 2014). However, blood doping was banned by the International Olympic Committee (IOC) in 1985, though there was no specific test for it at the time (Milne, 2006). Given the benefits of blood doping, many coaches and athletes still are trying to find ways to improve athletic performance in a similar fashion but through legal means. Compared with blood doping, the function of “live high, train low” is to improve aerobic capacity legally and without harmful side effects (Mairbäurl, 2013). In principle, human and animals exposed to high altitude conditions for a period of time, such as living on a mountain or sleeping in a hypoxic chamber that can simulate high altitude conditions, can allow humans or animals to obtain physiological acclamations, such as increases in red blood cell volume, erythropoietin (EPO), and heart rate (HR) recovery (Naeije, 2010), thereby improving their aerobic capacity. In other words, they can perform the same intensity of exercise more efficiently when they return to lower elevations (Levine and Stray-Gundersen, 1997). A large number of studies have shown the benefits of high altitude training to human athletes (Wolski et al., 1996; Stray-Gundersen et al., 2001), which raises interests in the horse industry. There is some anecdotal evidence to suggest some success of high- 1 altitude training in horses. For instance, Canonero II, purchased for $1,200 as a yearling, was shipped from Venezuela where he had been racing and training at altitude, shortly before racing in, and wining, the 1971 Kentucky Derby (Hunter, 2010). However, there is limited evidence in the scientific literature proving that horses can benefit from the physiological changes of chronic altitude exposure. Thus, there is interest in exploring the role of altitude training in horse racing, which could bring some scientific insights for horse owners in the future. Given they are commonly used by elite human athletes, it is not surprising that “high-altitude” or hypoxic chambers have also been designed for horses (Stewart, 2013). However, testing of such chambers has been limited. This study was therefore designed using a high-altitude chamber with simulated low oxygen conditions to determine whether horses could be acclimated to a high-altitude chamber (HAC) and to monitor their performance utilizing Standardized Exercise Tests (SET) and official races. We hypothesized that horses could adapt to a high-altitude chamber and that using chambers could enhance performance. 2 CHAPTER 1. Literature Review Equine physiological and metabolic responses to exercise Horses have a unique ability to manipulate their metabolic states in response to different intensities of work and exercise. There is a wide range between resting and maximal HR, and release of red blood cells (RBC) stored in the spleen occurs quickly and results in a substantial increase in oxygen-carrying capacity. These physiological changes, amongst others, help them improve their VO2 36-fold from the resting state compared to their maximum, which is more than twice that of humans and dogs (Thomas and Fregin, 1981). The RBC play a vital role in oxygen delivery for aerobic respiration. The quantity of RBC available help to determine the oxygen-carrying capacity of the blood. Hence, evaluating the hematocrit (Hct), which is the percentage of the blood volume made up of RBC (Brun et al., 2000), is often done. Within the RBC can be found hemoglobin (Hb), an oxygen-transport protein, that contributes to bringing the oxygen from the lung to the tissues for further metabolism (Jaussaud et al., 1994). Therefore, Hb is one of the main parameters commonly evaluated during physiological tests of aerobic capacity. The spleen serves as a blood reservoir in various types of animals, such as seals, dogs, and horses (Hinchcliff et al., 2008). The primary difference between the horse and human spleen relates to storage function. The horse’s spleen is capable of storing one- third to one-half of the red blood cells from the system (Thomas and Fregin, 1981). Moreover, the spleen of the horse plays an important role during exercise. During strenuous exercise or other circumstances like hypoxia, the spleen can contract - induced by chemicals called alpha-agonists - and mobilize stored RBC to the systemic circulation, 3 thereby enhancing oxygen transport capacity (Borrione et al., 2008). Releasing splenic red cell reserves leads to raising systemic hematocrit from 35% at rest to 60–70% during maximal exercise (Poole and Erickson, 2011). The capacity of the splenic reservoir in the horse is much greater than that in the dog (Vatner, 1974). However, the human spleen has long been known to lack this capability (Shephard, 2016). Therefore, the horse can be considered to be a “natural doper,” and this is one of the factors contributing to their remarkable athletic ability. Blood doping Blood doping is an approach used to increase RBC and, in turn, improve athletic performance. Blood doping has often been performed in races, particularly those requiring an endurance capacity. The most commonly used types of blood doping include injections of the pharmaceutical erythropoietin (EPO), injections with synthetic chemicals that can assist with carrying oxygen (Vit B12, folic acid, etc.) (Plumb et al., 2016), and blood transfusions, all of which are prohibited under the World Anti-Doping Agency's (WADA) List of Prohibited Substances and Methods (Docherty, 2008). Blood doping scandals in sports While blood doping techniques can be useful in and were initially intended to treat anemia, these techniques were then manipulated by athletes to improve their performance in sport. There have been many doping scandals in sports competitions. Blood doping was used to improve athletic performance as early as the 1972 Olympic Games (Leigh-Smith, 2004). In 1987, U.S.A. skier Kerry Lynch admitted to blood doping. German and Austrian cross-country skiers have also been accused of using blood transfusion (Eichner, 2007). 4 Arguably, the most well-known example of blood doping involves Lance Armstrong, an American cyclist who had his seven Tour de France titles revoked (Sparkes, 2004). Blood doping can attenuate muscle fatigue and permit the muscle to do more work for longer - increasing muscle efficiency by improving the supply of oxygen for the exercising muscle (Wan et al., 2017), thus explaining the enhanced performance by Armstrong in an endurance event such at the Tour de France. However, it will not increase the maximum force the muscle can generate (Wigmore et al., 2006). Blood transfusion A blood transfusion is a direct, practical, and effective way to improve Hb and Hct concentrations prior to an athletic contest. Transfusions were popular in the 1970s and declined after the administration of EPO became popular (Giraud et al., 2010). Doping with transfusions can be classified as either homologous and autologous (Goodnough et al., 1999). With a homologous transfusion, blood comes from a matched blood donor – thus, the athlete can avoid the negative effects of detraining associated with venesection (Jones and Pedoe, 1989). On the other hand, potential infection diseases, such as hepatitis and AIDS, and the possibilities of transfusion reactions are great concerns for people (Goodnough et al., 1999). To avoid such issues, autologous blood doping can be performed by removing two units (a 3% change in Hct is equal to 1 unit) of the athlete's blood, storing the frozen blood, and then reinjecting it about a week prior to the athletic competition (Klein and Anstee, 2014). In order to restore the athletes' hemoglobin back to the baseline level and recover from the detraining effect of venesection, the blood donation needs to be performed at least eight weeks prior to competition (Chapman et al., 1996). Since the donor and recipient blood are identical in 5 autologous blood doping, this is safer than homologous transfusion (Jones and Pedoe, 1989). Blood transfusion’s physiological effects Many studies show that blood transfusion can increase Hb and Hct concentrations in athletes and patients. Brien and Simon (1987) described that autologous infusion of 400 mL of red blood cells to distance runners lead to significant increases in Hct concentration. Further, an increase in the performance of cross-country skiers was observed after 1,350 mL autologous blood was infused at 3 h and 14 days (Berglund and Hemmingson, 1987). Similar effects occur in horses. Hurcombe et al. (2007) reported clinical variables from 31 cases of adult horses with different types of anemia receiving blood transfusions from 1995 to 2005. In those cases, low PCV, low hemoglobin concentration, and hyperlactatemia were commonly observed before transfusion and improved after transfusion. EPO’s role in erythropoiesis EPO is a 30.4-kD glycoprotein hormone with four carbohydrate residues that stimulates erythropoiesis in bone marrow, thereby increasing RBC to allow the blood to have a greater oxygen-carrying capacity (Miyake et al., 1977). EPO interacts and combines with the EPO-R receptor that exists in erythroid precursor cells (Krantz and Goldwasser, 1984). EPO production mainly develops in the proximal tubular region of the kidney in adult mammals (Walter et al., 2009). Many inducers lead to erythropoietin synthesis such as cobalt chloride and androgenic and anabolic steroids (Walter et al., 2009). Additionally, oxygen content can also play a fundamental role in EPO regulation. For instance, a decline in oxygen in the kidney results in EPO production (Eckardt and 6 Kurtz, 2005). Furthermore, hypoxia-inducible factor (HIF) can activate the EPO gene transcription, thereby stimulating synthesis (Fried et al., 1957). Doping with EPO However, administering rhEPO to racehorses is controversial. Many so-called EPO products targeted for horses lack scientific proof and have questionable ingredients. The average serum concentration of EPO is between 0 and 9 mIU/ml in horses (Jaussaud et al., 1994; Fu et al., 2011). One study in horses showed no changes in RBC mass, Hb concentration, or Htc after injection of 30 IU/kg bwt rhEPO but reported small changes with 120 IU/kg bwt rhEPO, and indicated that a higher dosage of rhEPO might need to be given in order to see note-worthy changes in hematological parameters (Jaussaud et al., 1994). Another reason that doping with rhEPO has less or no effect on the horse appears to be the difference between horses' own EPO and rhEPO. Piercy et al. (1998) suggested that rhEPO induced anti-rhEPO antibodies developed that cross-react with endogenous EPO, thereby inhibiting erythropoiesis. Furthermore, too much EPO can result in safety issues such as Htc that is too high, and that could potentially lead to blood clots, red cell aplasia, heart attack, and death (Hung et al., 2014), given more rigid and stickier blood of horses, compared to humans (Stull and Lawrence, 1986). One anecdote showed that, of 14 horses administered rhEPO, 8 developed anemia after being treated, and five of those eight horses died (Geor and Weiss, 1993). In general, there is limited research regarding EPO doping in horses, and the effect of recombinant human EPO on horses is still in question. Though increasing RBC has been a goal of some in horse racing, doping with EPO is illegal and an unsafe way to boost horses’ performance. 7 Cobalt As a trace mineral, cobalt (Co) also is an important component of vitamin B12 and the formation of cellular components of blood. Cobalt plays an essential role in the ruminant diet by participating with rumen microorganisms to synthesize vitamin B12. Besides, cobalt deficiency is associated with ill-thrift, reproduction, and production issues in cattle (Neal and Ahmann, 1937) and sheep (Suttle and Jones, 1989). On the contrary, there is limited literature that describes cobalt deficiency in the horse. Horses only need a small amount to maintain daily function. The 2007 Horse NRC estimated a Co requirement of 0.05 mg Co/kg of dietary dry matter. Normally, healthy adult horses can obtain sufficient Co from their diet as they can synthesize and absorb vitamin B12 in the hindgut (NCR, 1989). Further, horses may remain in good health while grazing low-Co pastures where cattle and sheep have died. Thus, horses seem to tolerate lower dietary intakes of Co than do cattle and sheep (Filmer, 1933). Nevertheless, excessive amounts of Co are associated with serious cardiovascular issues, potential nerve problems, thyroid toxicity, and death (Mobasheri and Proudman, 2015; Kinobe, 2016). Dietary Co often comes from Co-containing supplements and Co salt (Smith and Loosli, 1957). Normally, vitamin and mineral supplementation do contain small amounts of Co and, at those concentrations, have no detrimental effect on horses (Additives and Feed, 2012). It is common to provide such supplements to heavy workhorses in order to enhance their energetic performance and efficiency (Kinobe, 2016). 8 Cobalt’s role in blood doping World Anti-Doping Agency (WADA) had banned hypoxia‐inducible factor (HIF) products that include inorganic ionic Co in 2017 (Skalny et al., 2019). However, injection with Hemoplex® and Hemo-15® containing Co are approved for clinical use in some areas including Australia, Canada, and Hong Kong, and some Co-containing oral supplements like Horsepower® are commercially available as well (Kinobe, 2016). As such, misusing Co from supplements as a blood-booster may be common and raises concerns in the racehorse industry due to the ability of Co to induce erythropoiesis to enhance aerobic performance and carbohydrate metabolism (Peansukmanee et al., 2009; Ebert and Jelkmann, 2014). Cobalt’s threshold concentration and elimination Testing for Co misuse has been done using inductively coupled plasma mass spectrometry and suggested thresholds for cobalt concentrations both in serum and urine have been proposed. The International Federation of Horseracing Authorities (IFHA; 2018) suggested urinary threshold was 0.1 mg/mL and plasma threshold was 0.025 mg/mL in horse racing. Moreover, research found that the ratio of cobalt to vitamin B12 in plasma rapidly increased and then declined quickly, and returned to near baseline over the next week with the administration of a vitamin B12/Co supplement (Ho et al., 2015). Additionally, the urine ratio stayed above 10 for about 18 days after CoCl2 administration (Hillyer et al., 2018). Kinobe (2016) indicated that it takes 42 to 156 h to eliminate a large volume of residue in a horse after administrating CoCl2, which is likely to be different than with an oral Co supplement. 9 Cobalt’s role in erythropoiesis It has been known that Co plays a critical role in erythropoietin (EPO) synthesis for more than a century. As Co is a hypoxia-inducible transcription factor, it can activate the hypoxia-inducible factor (HIF) pathway, thereby inducing EPO transcription and production, which can potentially increase RBC mass and improve aerobic capacity (Goldwasser et al., 1985). Besides, Co was more likely to operate with generating a hypoxic state in the kidney resulting in erythropoiesis (Nangaku and Eckardt, 2007). Increases in EPO by the use of Co supplementation is effective in producing an erythropoietic response in rats and humans (Shrivastava et al., 2008). Mobasheri and Proudman (2015) reported the illegal usage of Co doping with their horses in order to anecdotally enhance aerobic performance in horses racing in Australia. Knych et al. (2015) demonstrated no effect of doping with a single dose of CoCl2 on hematological parameters, specific aspects in EPO production RBC parameters, or HR. However, horses displayed pawing, nostril-flaring, muscle twitching and even cardiac issues after receiving intravenous CoCl2 (at dosages of 4, 2, 1, 0.5, or 0.25 mg/kg) weekly for five weeks (Burns et al., 2018). However, the mechanism of Co influencing RBC production is complicated. Multiple related treatments and conditions appeared to be required before Co maximally stimulated HIF and induced EPO concentration, such as hypoxia and the dosage of Co. 10 Blood doping detections Blood doping is commonly used as it is easy to perform and difficult to detect (Lundby et al., 2012). With numerous abuses of blood doping and cheating in athletic contests during the last 40 years, the issue should not be ignored. There are some traditional tests which directly monitor exogenous manipulating agents implemented by WADA. Those direct detections rely on standard laboratory procedures (Pottgiesser and Schumacher, 2013). A test for EPO was introduced at the 2000 Summer Olympic Games in Sydney. The test was based on blood and urine matrices by using blood screening and urine test. Additionally, urine testing is the only scientifically validated method for direct detection of rhEPO. A test for homologous blood transfusions was implemented at the 2004 Summer Olympic Games in Athens. However, testing for autologous blood transfusion is challenging (Lundby et al., 2012), and the WADA continues to explore ongoing anti-doping research. The WADA also launched the Athlete Biological Passport where indirect markers are evaluated to test all types of blood doping (Müller, 2010). In horses, a different approach has been proposed to monitor rhEPO. Indirect double-blotting or direct liquid chromatography-mass spectrometry (LC-MS) methods have been applied in horse racing (Bailly‐Chouriberry et al., 2010). However, the duration of effective detection is only 48 h. Moreover, the latest technique with gene profiling investigation through Serial Analysis of Gene Expression (SAGE) has been tested for long-term detection to prevent doping with artificial EPO abuse, which is more promising (Bailly‐Chouriberry et al., 2010). A direct detection method was discovered by (Lasne et al., 2005), and they use the blood screening by an enzyme-linked immunosorbent assay (ELISA) and urine testing by 11 characterization of the urinary EPO isoelectric profile. This approach also can detect the hyper-glycosylated form of this drug (darbepoetin alpha) with subcutaneous administration. Based on those findings, the detection methods are still continually developing and upgrading. In 2018, the Kentucky Equine Drug Research Council gave funding to an ongoing project to explore biological passports for horses by examining changes in gene expression following the administration of EPO. Heather Knych, of the Ken L. Maddy Equine Analytical Chemistry Laboratory at the University of California- Davis, conducted this study and looked at changes in gene expression following micro- dosing of EPO (Angst, 2019). Altitude training With increasing altitudes, the fractional concentration of oxygen stays the same (20.9%), but the partial pressure of oxygen decreases (West, 1996). Altitude training has two basic regimes, “live high, train low” and “live high, train high.” The difference between these two protocols is whether training occurs at low or high altitudes. The most popular training regime that many coaches endorse is “living high, training low,” which aims to maximize hypoxia-acclimation at a high altitude while training intensity remains elevated while still training at sea level (Levine, 2002). Compared with training at high altitude, training at sea level is more effective (Adams et al., 1975). Hypoxia associated with training at altitude will decrease the amount of work that can be performed. Levine and Stray-Gundersen (1997) suggested that living and training at altitude have not been proven to be advantageous compared with equivalent training at sea level. 12 Elevations in training Atmospheric hypoxia is the basis of altitude training. For every 305 m above sea level that is increased, the amount of oxygen in the air decreases by approximately 3%. Thus, less oxygen molecules are available per breath and this contributes to elevated ventilation rate and heart rate (West, 2002). Exposure to extreme hypoxia at altitudes above 5,000 m can lead to considerable deterioration of skeletal muscle tissue and altitude sickness, which jeopardizes athletic performance and health (Hackett and Roach, 2001). Therefore, in order to achieve the physiological benefits of altitude training, appropriate altitude needs to be taken into consideration. In humans, an optimal elevation for residing at high altitude for acclimation purposes is 2,100 to 2,500 m (Levine and Stray-Gundersen, 1992). However, limited literature is available regarding appropriate altitudes for horses. One horse study examining horses’ response to 3,800 m altitude showed decreases in velocity (Wickler and Anderson, 2000). Physiological acclimations in human and laboratory animals Extended exposure to high altitudes can result in numerous physiological responses and hematological changes such as increasing oxygen-carrying capacity and Hb and Htc concentrations, enhancing lactate threshold (Ventura et al., 2000), and increasing mitochondria adaptation and concentration (Jacobs et al., 2016; Sanchez and Borrani, 2018). These responses, in turn, can improve athletic performance at sea level (Sinex and Chapman, 2015). Furthermore, the acclimation of hypoxia occurs in multiple systems throughout the body, such as the skeletal, muscular, and cardiovascular and respiratory systems (Rusko et al., 2004). In addition, 2,3-diphosphoglycerate (2,3-DPG) in the RBC is associated with Hb liberating oxygen to the tissues, and increases in the 13 concentration of 2,3-DPG can decrease affinity thereby helping oxygen transport (Sohmer et al., 1979). By increasing EPO production, the 2,3-DPG level can be raised (Sandhagen, 2001). Thus, 2,3-DPG has been used to evaluate EPO status. Furthermore, changes in the gene transcription of muscle (Zoll et al., 2006) and mitochondria components (Ponsot et al., 2006a) were also observed in humans. The mechanism behind those changes is complicated. The hypoxia-inducible factor pathway plays a vital role in response to hypoxia. Due to the transcription of the HIF-1 gene, EPO production is stimulated, inducing vascular endothelial growth factor and nitric oxide synthesis, which influences the aerobic and anaerobic capacity of athletes (Stroka et al., 2001). Besides, Hoppeler et al. (2008) suggested that high-altitude training can promote the strength and endurance of skeletal muscle due to increases in the transcription of mRNA of HIF-1and stimulation of growth factors activity. However, some work has reported no effect on skeletal muscle respiratory capacity after six weeks of high-altitude training (Lundby and Robach, 2015). Some human studies also reported that there was no positive result on mitochondrial respiratory capacity (Ponsot et al., 2006b) or even a decrease in mitochondrial concentration after hypoxic training (Hoppeler et al., 2003; Sena and Chandel, 2012). Hypoxic acclimations occur in other animals as well, such as rats (Ge et al., 2002), birds (Carpenter, 1975), and dogs (Hsia et al., 2007) but there is relatively little literature pertaining to the acclimation of horses (Wickler and Greene, 2003). Can horses adapt to hypoxic environment? It is known that acclimating to a new environment is challenging for competing horses, whether the acclimatization is to different altitudes, time zones, or other inevitable 14 factors (Sellnow, 2006). However, exposing horses to high-altitude conditions has a long history. Since the age of Genghis Khan, Mongolian horses have been used for military operations on the plateau (Weatherford, 2005). In present times, some horses, like Tibetan horses and Mongolian horses, are still living in the mountains and high-altitude areas (Xu et al., 2007). Moreover, horses are used to carry and transport goods in the mountains, and also for recreational riding, which indicates that they are capable of adapting to conditions that are lower in oxygen. Research indicated increased altitude accompanying elevated heat and humidity did not lead to the adverse effects on equine athlete’s capacity and other physiological condition during 3-day eventing in Colorado (1,900 m above sea level) (Foreman et al., 1999). In addition, increases in PCV were found when horses were transported to Mexico City (2,240 m) for races, indicating horses may adapt to higher altitudes (Sellnow, 2006). Duration of hematological acclimations Acclimation is not instantaneous but can develop relatively quickly. From human studies, increases in EPO can be found within a few hours after exposure to altitude. The peak of EPO concentrations occur within 2 to 3 days following initial exposure to altitude and slowly returns to baseline in 14 to 28 d (Sinex and Chapman, 2015). RBC counts and Hb concentration can increase dramatically after a couple of weeks at high altitude, and RBC volume is reduced quickly while returning to sea-level due to neocytolysis, which involves reducing RBC count primarily through the destruction of young RBCs (Merino, 1950). In all, several studies showed that the endurance of blood value acclimation is 2 to 3 weeks (Heinicke, 2005; Rodríguez et al., 2007; Brocherie et al., 2015). 15 Previous studies in horses While studies conducted in horses evaluating the effects of high-altitude acclimation are few, some have been done. Wickler and Anderson (2000) examined changes in hematological parameters and athletic performance in 6 horses in response to 9 days at high altitude (3,800 m above the sea level). The results showed that RBC and DPG/Hb concentrations increased shortly after returning to sea level and the horses had faster heart rate recovery and lactate recovery as well. Furthermore, the endocrine responses of the same horses were evaluated at the same altitude, but at different time periods. Just one day after arriving at high altitude, there were significant increases in glucose, cortisol, and thyroxine levels, but they return to normal within the following days. Bicarbonate ion, a gas change indicator, also returns to normal within the first 3 days at altitude. The changes indicate that horses could acutely acclimate within 2 to 3 days to such an altitude (Greene et al., 1999; Greene et al., 2002). High-altitude chamber With the development of technology, high-altitude chambers have been designed and can play an essential role in altitude training. Athletes can naturally expose themselves to a high-altitude environment by spending extended periods of time daily at a location at a high altitude such as on a mountain. Moreover, athletes can also use advanced altitude chambers that mimic the high altitude environment up to 5,500 m by providing hypoxic air, which can effectively induce acclimation and enhance performance (Levine and Stray-Gundersen, 1992). For instance, a Finnish scientist named Heikki Rusko designed a “high-altitude house”. By changing the concentration of oxygen to about 15.3%, a high-altitude environment can be mimicked (Faiss et al., 2013). 16 Athletes can live and sleep inside the house but perform their training outside with normal oxygen concentration (20.9%). Several companies mimic hypoxia using a normo- baric artificial atmosphere (Richard and Koehle, 2012). There are various types of chambers, ranging from specialized training rooms to bed tents. Many environmental parameters can be controlled, such as humidity, temperature, and “evaluation” by the hypoxic systems. These chambers for humans are relatively easy to get access to and are becoming more effective and safer. Though rare, altitude training systems for horses have been developed. For instance, racehorse trainers in Japan and Australia have applied hypoxic training to their horses. Moreover, the maiden racehorse Shamus Award won the Cox Plate (Group 1) in Australia with a AUS$3 million purse and it was suggested that horse was being stabled in such a “high-altitude” chamber (Stewart, 2013). While altitude training is a growing industry, the scientific support has still been questioned (Winther, 2018). Previous studies and information indicate horses are capable of naturally acclimating to high altitudes, however, this study aims to determine whether horses could also adapt to an advanced facility in which a “high-altitude chamber” is used to affectively simulate hypoxic conditions found at altitude. We aim to explore some physiological adaptations in those horses and possible effects of the chamber on Thoroughbred race performance. This project was a pilot study and designed to provide scientific insights for the sport horse industry. 17 CHAPTER 2. Effects of “High Altitude Chamber” on Racing Thoroughbreds Materials and methods The project was approved by the Michigan State University Institutional Animal Care and Use Committee via an exemption approved on June 15, 2018. Design of high-altitude chamber. Prior to the start of this work, the owner of the Thoroughbred facility retrofitted a room in his barn to make into a HAC. The HAC had to be air-tight, have an additional power supply, and be equipped to produce hypoxic conditions. Equipment was housed outside the HAC and included a rotary screw air compressor with tank and dryer (Aircenter SK15, Kaeser Compressors, Inc., Fredericksburg, VA) to make incoming gas compressed and flow into the Simulated Altitude Generator (Hypoxico Altitude Training Systems, Hypoxico, Inc., New York, NY). This generator provided oxygen-depleted air to a stall having ventilating openings for equivalent pressure inside. The system contained digital internal and external monitoring systems and could control oxygen concentration from 20.9% to 9%. The inside chamber contained two separated stalls (3.1×3.1 m) equipped with sealed door and walls, hay bags, water buckets, oscillating fans, air conditioning, and scrubber machines to remove CO2, methane, ammonia, and excess water produced by horses. Moreover, there was an ammonia monitor and oxygen sensor to ensure horses’ safety. The HAC was equipped to have an emergency ventilation door open if conditions inside the chambers were to exceed safety margins. The chamber temperature was approximately 18℃ in the winter and 21℃ in the summer. Stalls were equipped with a drainage system to allow for urine removal. Feces were manually removed every 12 hours. Cartridges for CO2 scrubber were replaced every 36 h. 18 Experimental design. The experiment was divided into two trials. Trial 1 lasted two months and involved two 4-yr-olds Thoroughbreds, one gelding and one filly, with at least 6 prior racing starts that were housed in the HAC to test the adaptation of the horses and chamber equipment. All horses in study were owned by, and under the management of, a Thoroughbred breeding farm and racing facility in Ohio. The horses were in healthy physical condition as needed for racing. Taking into consideration the horses’ safety and health, oxygen level was gradually decreased daily to target setpoint (13.5%) that was reached after a two-week period to allow horses to adapt to low oxygen conditions. On a daily basis, within the hypoxic system, it took almost 1.5 h to decrease the oxygen concentration from 21% to 18% and then approximately an additional three hours to reach the setpoint 13.5% (simulated altitude at 3,200 m). Thereafter, horses stayed at the HAC with 13.5 % oxygen content for 8-10 h/d. All horses were provided with ad libitum access to water and grass hay. When out of HAC, horses stayed in traditional box stalls and were turned out in a paddock if weather permitted. Additionally, horses were conditioned with 30-min training on treadmill including walking, trotting, and cantering six days per week. On d 37, the horses were sent to a racetrack, at which point their daily exercise was on the dirt track with a rider. After 7 d, they returned to the farm and resumed daily HAC occupation and both track and treadmill exercise through the completion of trial 1 except when transported to the racetrack for racing. Horse 1 raced on d 62 (one day after the trial ended) and Horse 2 raced on d 41 and d 56. Resting Hb was measured on d 0, d 37 and at the end of the trial (d 61). Trial 1 began in September 2018 and ended in November 2018. Trial 2 began in December 2018 and finished in January 2019. Two 2-yr-olds 19 Thoroughbreds fillies, with a minimum of 3 prior starts, were stalled in the HAC, with the oxygen level gradually decreasing to 13.5% during the two-week adaptation. They stayed with a 13.5% oxygen level for 8-8.5 h/d. When out of HAC, horses were maintained in normal box stalls on the farm. Horses were conditioned on the treadmill or track 6 d/wk. When not provided controlled daily exercise, horses were allowed turnout in a paddock. Conditioning regimen consisted of 30-min of exercise including walk, trot, and canter on a high-speed treadmill (Chadwick Engineering Ltd., Canada) or with a rider on a dirt track, consisting of a 15-min walk (warm-up), a 4-min trot, a 4-min canter, a 2-min gallop and then 15-min walk (recovery). The choice as to whether the exercise was performed on the treadmill or track was weather-dependent and made by the resident trainer and was consistent with the work being received by other Thoroughbreds in race training at the facility. During the trial, the horses were shipped to a track on day 22 for 5 days and then reentered the HAC for the remainder of trial 2 except when transported to the racetrack for racing. Horse 3 raced two days after trial 2 ended and Horse 4 raced 8 days after trial 2 ended. Horses received race training on the dirt track with rider during the time between the end of the trial and racing. In this trial, Hb was evaluated immediately upon cessation of SET. Besides maximal HR, the HR at 3 min and 5 min post-exercise was recorded on day 0, day 10, and day 31. Exercise test. The SET was performed on the treadmill (Chadwick Engineering Ltd., Canada) outside the HAC, consisting of walking 1.6 km at 1.8 m/sec with an 8% slope, trotting 1.6 km at 3.4 m/sec with 3% slope, and walking 0.8 km at 1.8 m/sec. Due to equipment limitations, the SET did not include canter exercise The SET was performed at day 0 and repeated on day 10 and 31. 20 Iron (Fe) supplementation. Before the study started, the first two horses were administered 200 mg/day Fe orally for four weeks. However, supplementation was ceased during the study. Sample collection. Blood samples were taken via jugular venipuncture with vacutainers (BD Vacutainer®, Becton Dickenson, Franklin Lakes). Samples were placed in refrigerated bags, transported immediately after collection to the MSU laboratory, where whole blood was used to analyze Hb levels following the procedure of Stan-bio (HemoPoint® H2). Blood samples for iron analysis were centrifuged at 3,500 x g for 20 mins. The aliquots of serum were stored at -20° C until analysis. Serum iron was determined by a colorimetric method (Wako Pure Chemical Industries, USA) with an automated analyzer. TIBC was calculated, following the manufacturer’s instructions. The equation follows as: TIBC (μg /dl) = (Aunk – Ablk) / (Astd – Ablk) × 400 Where Aunk, Ablk and Astd are the absorbance of the plasma unknown, blank, and standard. HR was measured by heart rate monitor (Polar, Bethpage, NY) during maximal speed, and then at 3 min and 5 min post-exercise. Performance evaluations. All horses’ race results were obtained from Equibase (https://www.equibase.com) and average finishing position within a race and the average speed ratings from races before starting the trials were compared to the races after commencing the trials. 21 Statistics All data from the SET trial are presented as mean ± standard error. Hemoglobin concentration was assessed by ANOVA in the proc MIXED program of SAS version 9.0 (SAS Inc., Cary, NC, USA). The model is the fixed effects of the day. HR at maximal speed, 3 min, and 5 min post-exercise were compared using a paired Student’s t-test to assess performance differences based on the physiological changes that occur with acclimatization. Significance was defined as P≤0.05. Results Iron profile after 4-wk supplementation before the study. Table 1 shows the serum iron and TIBC of the first two Thoroughbreds in this study. The standard ranges of serum iron and TIBC are 53-209 μg/dL and 244-480 μg/dL, respectively (Assenza et al., 2016). Table 1. Serum iron and TIBC in the first two Thoroughbreds after 4-wk supplementation before the study. Horse Serum iron (μg/dL) TIBC (μg/dL) 1 2 124 119 404 416 Resting Hb in trial 1. Table 2 illustrates the resting Hb measured on day 0, day 37 and day 61. The standard range of resting Hb is 12 to 18 g/dL (Stewart et al., 1977). 22 Table 2. Resting Hb (g/dL) of two Thoroughbreds in trial 1. day 0 14.9 15.2 day 37 14.8 15.1 day 61 15.1 15.2 Horse 1 Horse 2 Hb level in trial 2. Table 3 illustrates that the Hb measured immediately post- exercise was significantly greater on day 31 (17.0 g/dL) compared to day 0 (16.0 g/dL, SEM=0.1; P<0.01). Table 3. Hemoglobin concentration (Hb) in two Thoroughbreds after SET on day 0, 10, and 31. Hb, g/dL Horse 3 Horse 4 Average day 0 15.8 15.9 15.9a day 10 15.9 16.1 16.0ab day 31 17.1 16.9 17.0b SEM=0.1, P Value=0.005 ab Means not sharing similar superscripts differ (P < 0.05) 23 Figure 1. Means of Hb on day 0, 10, and 31. Means not sharing similar superscripts differ (P < 0.05) ) L d / g ( e v e l b H 17.5 17 16.5 16 15.5 15 14.5 b a ab day 0 day 10 day 31 HR in trial 2. The average maximum HR measured on d 0, 10, and 31 during the SET was between 110 and 120 and did not differ between days. Likewise, HR at 3 minutes post -SET ranged from 61 to 68 (SEM=2, p=0.18) and HR at 5 minutes post-SET ranged from 54 to 63 (SEM=7, p=0.64). No differences in HR were seen between days (Table 4). Table 4. Average HR in two Thoroughbreds after SET on day 0, 10, and 31. Average HR, bmp day 0 day 10 day 31 Max 120 110 120 3-min post SET 5-min post SET 68 68 61 63 62 54 24 Race performance in trial 1 and 2. Table 5 shows the average race results from all the four horses studied in the two trials – contrasting their performances after HAC to their races prior to commencing this study. As can be seen, no improvement in the average finish position was noted and the average speed rating decreased in the races performed after HAC adaptation compared to prior to HAC adaptation. 25 Table 5. The average finish position and average speed rating score both prior to HAC acclimation and after HAC acclimation in four Thoroughbred racehorses along with the number of races from which the average was determined. Trial 1 Horse 1 Average finish position (# of races) Prior HAC After HAC Horse 2 Prior HAC After HAC Horse 3 Prior HAC After HAC Horse 4 Prior HAC After HAC Trial 2 6 (6) 5 (1) 6 (6) 9 (2) 4 (5) 5 (1) 5 (3) 5 (1) 26 Average Speed Rating 52 41 23 17 36 14 31 2 CHAPTER 3. Discussion and Conclusion Discussion: Incomplete limited characterization of athletic performance, lack of controls, and small subject numbers have complicated the interpretation of this study evaluating the use of a HAC. Changes over time are confounded with potential training effects (Soroko et al., 2019). Despite these major limitations, this work can provide some insights into the acclimation of horses in a HAC. Behavior. While not specifically measured, changes in behavior were noted throughout the study. In trial 1, when the horses were first exposed to 13.5% oxygen, they remained sedentary and just stood in a corner with their heads down – suggesting they were possibly experiencing altitude sickness such as occurs with humans (Moore and Regensteiner, 1983). However, after two weeks of exposure to low oxygen, behavior appeared to return to normal. The return to normal behavior suggested that horses could adapt to a HAC but that adaptation was not immediate and horse welfare needs to be considered. Hb level. The interpretation of resting hematological parameters can be somewhat ambiguous. This is because the horse’s spleen is capable of storing one-third to one-half of the RBC (Poole and Erickson, 2011). The spleen can make active contractions induced by chemicals called alpha agonists and mobilize stored RBC to the systemic circulation in response to varying stressful stimuli, such as hypoxia. Releasing splenic red cell reserves (at 80 to 90% hematocrit) leads to raising systemic hematocrit from 35% at rest to 60 to 70% during maximal exercise (Poole and Erickson, 2011). In trial 2, Hb concentrations were increased after HAC exposure. Similar results were also reported 27 among previous human and other animal studies (Heinicke et al., 2003). Hb concentration is a favorable indicator that supports aerobic performance. However, without a control group, we cannot identify the effect of the HAC that attributes to changes in blood parameters since strenuous exercise and training could potentially boost blood values as well (Soroko et al., 2019). Iron supplementation. Iron plays a role in enhancing Hb concentration and the activity of RBC. Exercise can induce changes in iron metabolism to mimic iron deficiency, thereby decreasing Hb and ferritin concentrations in humans. Iron supplements are often used by elite athletes during extreme exercise (Beutler, 2002; Zoller and Vogel, 2004). In human, Govus et al. (2015) suggested that sufficient iron stores are required to support increased erythropoiesis during prolonged altitude exposure. Moreover, a three- to five-fold increase in erythropoiesis occurs and was associated with 100% erythroid iron uptake during the first few days of adaptation. Recognizing this, the racehorse facility owner supplemented the two initial horses with Fe to elevate their Fe status to facilitate erythropoiesis. However, TIBC and serum iron value are similar to Assenza’s study of horses with standard training program (Assenza et al., 2016). Some studies have reported increased serum iron concentrations in horses following exercise, probably resulting from the release of iron from reticuloendothelial cells (Assenza et al., 2016; Assenza et al., 2017). Iron metabolism can be influenced by the duration and intensity of exercise (Assenza et al., 2016). However, it is unusual for horses in training to have an iron deficiency. Typically, there is no need to provide additional iron supplements for horses due to an abundance of iron in horse’s diet (Richards and Nielsen, 2018). 2007 Horse NRC 28 suggested Fe 40 ppm for adult horses. Massive iron may result in depression, dehydration, and diarrhea (Klosowicz, 2017). At this time, there is no indication that iron supplementation is needed or beneficial to racehorses and the owner, after seeing the initial results from the horses in trial 1, decided to not continue with Fe supplementation in trial 2. Fluctuation of oxygen level in HAC when horses enter or leave. When opening the door of the HAC to lead a horse through, air within the HAC is allowed to mix with air from outside the HAC resulting in small changes in oxygen concentration (2-3%) that were noted for 15 to 20 minutes before returning to the setpoint. However, with the oxygen sensor near the door, it is unlikely that the oxygen concentration of the whole room was altered much during those brief periods. Regardless, these brief alterations were noted as a reminder that any time the door is opened, there is a period in which the gas concentrations are altered before homeostasis is once regained. Environmental factor of HAC. Conditions of air quality and sanitation in the HAC can have a direct effect on the health and performance of horses. Humans sleeping in a hypobaric chamber or high-altitude tent experience different environmental conditions than would a horse residing in one. Air pollutants originate mainly from their feces, urine, feed, and bedding. Moreover, horses undergo hindgut fermentation – resulting in carbon dioxide, methane, and ammonia being released from horses – gases which should be removed from the chamber. Horses can potentially produce 20.7 kilograms of methane gas (Elghandour et al., 2019) and 12.2 kilograms of ammonia (EPA, 2004) per year. From this standpoint, filter systems are a critical part of the insulated chamber to remove those biochemical wastes and keep the airflow in the chamber clean and fresh. At the time of 29 this study, to purchase cartridges to remove CO2 for the chamber, the cost would have been around $350 and the cartridges would need to be replaced every 36 hours - resulting in high operating costs. Beyond gas production, horses can produce large amounts of sweat and also release substantial moisture through their respiration. After racing poorly, one of the horses in trial 2 had an endoscopic evaluation done. An accumulation of mucus within the airways was found and it is believed the high humidity in the chambers likely contributed to mucus production and secretion (Gerber, 1973). This likely had a negative effect on racing performance. Suggestions and implications from HAC. As a pilot field study, we confronted various unexpected challenges with using the HAC. While this study was not able to demonstrate improved performances with the use of HAC, the findings are useful in providing guidance to others that may be interested in exploring work in this area. Using the behavior of the horses as a guide, lowering the oxygen concentration in the chambers needs to be done over two to three weeks to allow adaptation without compromising animal welfare. When the oxygen concentration was dropped too quickly after initially placing the horses into the HAC, a lack of voluntary movement in the chambers suggested adaptation had yet to occur. As horses seemingly adapted to the HAC, their behavior returned to normal. While this study was originally designed to evaluate physiological acclimation, the behavioral adaptation should also be taken into consideration. In terms of oxygen level, a 13.5 % concentration, similar to Wickler’s study (2000), was targeted. The initial assumption was that an ideal concentration that could be 30 determined and that concentration could be used for a larger study with more horses. Towards the end of the two-week adaptation period in trial 1, the oxygen concentration was temporarily dropped to 13% but the behavior of the horses was concerning (they appeared to be abnormally depressed and lethargic) and the concentration was returned to 13.5% at which point the horses resumed eating and normal behavior within the HAC. Again, this study serves as a reminder that even if improvements in athletic performance were gained, the well-being of the horses needs to always be considered. Adaptation needs to be gradual and an ideal oxygen concentration for an equine HAC still needs to be elucidated. The ultimate goal of this study was to determine if the use of a HAC could improve athletic performance in horses. While our study did not confirm such improvements, it did reveal potential concerns with using such a system. Beyond needing to remove large quantities of gases such as carbon dioxide, methane, and ammonia, high humidity within the chambers may have been the cause of the excess mucus found in the airway of one of the study horses that had an endoscopic evaluation after a poor race performance. These are not issues that would be present to a great degree with human HAC but are challenging to deal with in an equine HAC. In addition to the gas removal, a dehumidifier should be used to keep moisture within the chambers to an acceptable level. While a HAC allows oxygen concentrations to be controlled allowing horses to “live high, train low” without actually living at a high altitude, there are many limitations. Using a HAC is time-consuming and installing and operating the system is expensive. While there is vast experience using such systems with humans, many modifications need to be made to a HAC system to allow it to be used for horses given that horses excrete 31 waste while in the chambers and this waste must be removed promptly. Besides the high cost and large investments of time needed to manage horses in the chambers, horse welfare must be considered – particularly during the initial adaptation stage as horses are becoming acclimated to decreasing oxygen concentrations. Conclusion: Although limitations existed in this pilot study, the findings of this study demonstrate that horses could adapt to a HAC, but this process takes time. Also, by not having control horses housed outside the HAC, any increases in Hb that may be associated with HAC use were confounded with a potential effect of training. While having control horses should be the standard for any research project, this rare opportunity to study horses in a HAC and undergoing actual racing provided some knowledge that could be used in further controlled studies. Further, this preliminary work showed no improvements in finish position and speed rating after HAC. HAC is a high cost and time-consuming investment and any desire to invest in such should be taken only after great consideration. Whether HAC could enhance horse race performance is still in question. 32 REFERENCES 33 REFERENCES Adams, W. C., E. M. Bernauer, D. Dill, and J. B. Bomar Jr. 1975. Effects of equivalent sea- level and altitude training on VO2max and running performance. J. Appl. Physiol. 39:262-266. Assenza, A., S. Casella, C. Giannetto, F. Fazio, F. Tosto, and G. Piccione. 2016. 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