PHYSIOLOGICAL RESPONSE TO EXERCISE AND TRAINING IN HORSES: EXPERIMENTS AND STUDIES BENEFITING HORSE AND HUMAN HEALTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1019256
• Project Start Date: Apr 1, 2019
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559

Performing Department
Animal Sciences

Non Technical Summary
Papers published from the Principal Investigator's work at Ohio State University and from data generated at the Rutgers Equine Exercise Physiology Lab have documented the ergogenic (performance enhancing) and ergolytic (decreased performance) effects of several drugs and practices. Some of the projects published by the PI that have examined the effects of drugs have included studies of the effects of furosemide, phenylbutazone, cocaine, epogen (rhuEPO), equine somatotropin, cobalt infusion, E-4021, exogenous endorphin, glucose infusion, tryptophan administration. More recently the investigator has examined the effects of clenbuterol administration and various alkalinizing agents ("milkshakes"). These studies just touch on some of the substances, both legitimate therapeutic drugs and illicit agents, that are given to horses. Data from the investigator's papers have been used to form policy decisions about some of these agents; however, more studies are needed to examine the effects of therapeutic agents commonly used to treat horses to determine if they have any effect on markers of performance. During the past five years the PI has received substantial funding from the pharmaceutical industry to conduct pharmacokinetic and pharmacodynamics studies of a novel type 5 phosphodiesterase inhibitor that is being tested for FDA approval. The current project will continue this work.Most of the externally funded research conducted by the PI has been focused on food extracts that reduce inflammation . We have published studies of the effects of cranberry, orange peel, black tea, and ginger extracts and have documented that cranberry and tea reduce markers of exercise-induced inflammation in blood. We have also documented that orange peel and ginger reduce the time needed for recovery from intense exertion. The lack of information whether other nutraceuticals affect exercise capacity is a concern to regulatory agencies.The second objective of this project will examine the effects of nutraceuticals on the horse.The PI and his students have published a large body of information on the effects of aging on the ability to perform exercise and the effects of training on the reversal of various aging-induced changes. Recent studies in the lab have focused on the effects of aging and training on the hormones associated with energy homeostasis (insulin, leptin, adiponectin, cortisol, ghrelin, etc.) with a focus on why training disrupts the drive to eat and. The PI, his collaborators, and students, are currently using the horse as an animal model to investigate aging-related alterations in the control energy balance. Collaborative studies with investigators from multiple universities has yielded information on the role of the inflammatory cytokines in the process some call "inflamm-aging". Current studies are examining the effects of aging on signaling proteins important in the control of glucose homeostasis during and after exercise. The data will shed new light on how aging (and a lack of exercise) results in obesity and insulin resistance in the horse and humans.Another area of ongoing research involves the PI's collaboration with investigators from Nutritional Sciences. We are currently examining the effects of training and detraining on body composition and protein metabolism with a special focus on signaling proteins and pathways related to amino acid and protein metabolism. The PI and collaborators are also using novel tools, like metabolomics, to examine potential mechanisms associated with the changes in muscle mass seen with chronic exercise. That work will continue over the next few years and 4 papers are currently being written.Finally, the PI and his graduate student from Iraq have conducted studies looking at the effects of acute exercise and exercise training on the microbiome. These studies have produced three papers and another is on the way. Those efforts have resulted in the first papers published on the effects of acute and chronic exercise on the microbiome of the horse. This collaborative research has involved investigators from the University of Delaware and the Rutgers Department of Microbiology and Biochemistry. We will continue to explore the effects of various management practices on the microbiome as well as other aspects of horse health.Lastly, the PI has been involved in projects focused on emerging areas related to the physiology of the horse. One collaborative study examined the effects of equine assisted activities on biomarkers and heart rate variability in the horse. The horse human bond is an emerging area of research and the PI will continue to collaborate on projects that focus on the physiological responses and adaptations seen in these horses. Another emerging area of research being explored by the PI is the development of evidence approaches to modify training programs in non-racing breeds including polo ponies. The PI has facilitated planning with investigators from Argentina and the University of Maine. This collaborative international effort will yield information gained in the lab that can be applied to real life utilization of physiological

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3810	1020	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1020 - Physiology;

Keywords

horse

equine exercise

equine exertion

pharmaceuticals

nutraceuticals

microbiome

aging

Goals / Objectives
Objective 1: To continue to perform a series of experiments to determine if various exogenously administered therapeutic agents will alter aerobic capacity and exercise performance in horses.Objective 2: To continue to perform a series of experiments to determine if various exogenously administered nutraceutical agents will alter aerobic capacity and exercise performance in horses. In particular, to examine the uptake and disposition of flavanols into blood, muscle, and urine and to determine if they reduce cytokine expression in muscle biopsies.Objective 3: For this objective, studies will be conducted to further investigate the physiological mechanisms associated with the effects of aging and training on the endocrine response to exercise with a special focus on the control appetite and energy homeostasis.Objective 4: Examine the effects of exercise on emerging areas of interest, for example the microbiome, metabolomics profiles, heart rate variability and autonomic balance, and the development of new training paradigms for non-racing breeds of horses such as polo horses, the horse human bond.

Project Methods
Studies to Address Objective 1:Various therapeutic drugs currently in use by veterinarians in the Northeastern United States will be studied. Thus, data gained may be used by a multiple state racing commissions and the governing bodies of the eventing groups (EUSA, USET, AQHA, FEI, etc.). Drugs to be chosen will include antibiotics, vaccinations, acid therapy agents (omeprazole, rinatidine), beta-agonists (albuterol, clenbuterol, salmuterol, etc.), local anesthetics (lidocaine, carbocaine, etc.), cobalt salts, E-4021, and other drugs either found to be a problem in racing.Animals: All methods and procedures will be performedwith the approval of the RU ILACUC.nstitutional Twenty, clinically healthy, Standardbred mares (-450 kg; 4-9 yrs) will be used. The mares will be unfit, but accustomed to the lab and running on the treadmill prior to the start of the experiment. During the trial the horses will be housed as a group on a pasture.General Experimental Protocol: The individual drug experiments will be conducted using a series of incremental exercise tests (GXT) and simulated race exercise tests (SRT) to evaluate the effect of various therapeutic agents on markers of performance. The studies will be conducted using a crossover design so that each horse is tested after receiving either the placebo control or drug. The experiments will involve conducting the first GXT on one day and the first SRT two days later. Two weeks will be allowed between the first SRT and the second GXT to prevent any carry-over training effect and to logistically maximize the use of the treadmill lab. The drug treatments used will be administered approximately 6 hrs prior to the exercise test (GXT or SRT). Horses will then be brought into the treadmill barn and prepared for the GXT or SRT. Pre-exercise jugular blood samples (20 mL) will be collected into tubes containing lithium heparin. Samples will be collected before giving the supplement (-6 hrs) and at ten min before the test and stored on ice until analyzed. After this, horses will undergo the GXT or SRT followed by post-SRT blood samples collected at 60 and 90 min post-exercise (also stored on ice). The process will be repeated for each drug/control pair.Exercise Test Protocol: At the beginning of the experiment each horse will undergo an incremental exercise test (GXT) to measure maximal oxygen uptake (VO2max), blood lactate concentration, hematocrit, and plasma protein concentration. These tests will be conducted using previously published techniques (17-24). Measurements and blood samples will be obtained before exercise, for each step of the GXT and at 2 and 5 min after the GXT. Data will be used to calculate the speed and watts atVO2max, at a blood lactate concentration of 4 mMol/L, and at the maximal step of the treadmill test. The velocity to produce VO2max and the VO2 speed relationship will be used to establish the individualized speed to be used in the simulated race tests (SRT).During the SRT, horses will stand quietly for a 10 min equilibration period. The pre-exercise blood sample (20 mL) will be obtained at the end of this equilibration period. The mares then will run on the treadmill at a fixed 6% grade for a 2 min warm-up at 5 mis, followed by a 2 min run at a speed calculated to be 120% of the speed needed to elicit VO2max, and then for an additional 2 min at 5 m/s as a cool down.Studies to Address Objective 2: Drugs to be studied: Various nutraceutical agents, currently in use by horse owners and veterinarians in the Northeastern United States will be studied (various herbal supplements, vitamin/mineral/amino acid mixes, etc.). Methods and procedures will follow the outline provided above for Objective 1.Studies to Address Objective 3: The effects of aging and training will be conducted using the acute exercise tests outlined above and exercise training as performed in several published studies (3, 47-49). Specific hypothesis driven experiments will be conducted with the appropriate designs so as to determine the effects of aging on physiological function and to determine if chronic exercise (training) and can ameliorate some of those alterations physiological function associated with aging. In our past studies we have demonstrated that acute exercise affects the cytokines and myokines that modulate the glucose and insulin response to exertion Studies in this section will be designed to determine the effects of training and if it can reverse aging-induced alterations in the endocrine control of energy homeostasis. Planned studies will focus on the measurement of signaling proteins that may explain how training can modulate aging-induced alterations in muscle metabolism.Studies to Address Objective 4: Research efforts by the PI have been those of an investigator who uses exercise and training as a major physiological perturbation to evoke acute and adaptive integrative systems responses that may be important for the understanding of the athletic horse. Objective 4 takes the tools and protocols used for objectives 1-3 and applies them to emerging areas of research.The PI conducted the first studies of the effects of exercise and training on the microbiome. We will advance that line of research by exploring the effects of various management practices on the microbiome.There are many unanswered questions associated with how we care for horses and how those practices affect the microbiome. We will address a few of those questions related to the athletic horse. For example, furosemide is a diuretic that is given to 70-90% of the horses that are racing. Past studies have shown that furosemide can cause up to 25 kg of urine loss prior to a race with substantial fluid, electrolyte, and acid-base disturbances. We will conduct experiments using the acute exercise tests outlined to test the hypothesis that furosemide can alter the microbiome of the horse.Another common practice is the nasogastric administration of alkalizing agents or "milkshakes" to horses to enhance performance. These substances buffer the metabolic acidosis that occurs with high intensity exercise. In its simplest Racing commissions have established testing thresholds that rely on the measurement of physiological variables such as plasma total carbon dioxide concentration (tC02). We will conduct studies to examine the effects of milkshake administration on the microbiome of the horse.We have recently published a paper documenting the effects of equine assisted activities on markers of stress and autonomic balance in both the participating horses and the humans. We will continue to conduct studies to examine the effects of training status, age, feeding practices and other variables that have the potential to influence the physiology of horses used in equine assisted therapy and equine assisted activity programs. As with our published study, will partner with investigators in multiple departments and at multiple institutions to collect data from horses at a number of off campus centers conducting Equine Assisted Therapy and Equine Assisted Activity program.Finally, another emerging area of work being explored by the PI is the development of new methods to assess training status of non-racing breeds of horses. The PI presented an invited lecture "From the Laboratory to the Track: Training Horse" at the 2nd Simposio International de Nutricion Animal de Biofarma in Rosario, Argentina in 2018. Key members of the Polo industry in Argentina asked the PI to help develop new methods to monitor training and to adjust training protocols in elite polo horses. The PI has established a preliminary working group with the head veterinarian from Argentina's best polo center and investigators from the University of Maine. This preliminary multi-institutional and multinational work will be developed further research with possible inclusion of studies conducted in the laboratory that can be compared to data collected in the field.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audiences reached by the efforts for the efforts this year include students, faculty, colleagues in the field, horse owners, trainers, veterinarians, regulators, legislators, and anyone interested in the health and well being of horses. Changes/Problems:COVID-19 has brought all of our research to a halt during the spring and summer periods. What opportunities for training and professional development has the project provided?Two PhD students, one Masters Student, and a visiting scientist from Brazil collaborated in all of the projects. How have the results been disseminated to communities of interest?Results have been diseminated in published papers and lay and professional talks. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue to conduct studies to generate new information related to the goals stated above.

Impacts
What was accomplished under these goals? We conducted a number of experiments. One examined the effects of clenbuterol administration on body composition; a second examined the effects of clenbuterol on force of muscle contraction during exercise;a third examined the effects of furosemide and exercise on the microbiome'a fourth examined the effects of furosemide and exercise on the inflamatory cytokines and cortisol response to intenseexercise; a fifth tested a wearable IMU-based early limb lameness detection for horses using multi-layer classifiers; a sixth examined the effects of various psychological stressors on muscle EMG and cortisol responses in horses. One study seeks to determine if cobalt may actually act as a performance enhancing drug (PED) by altering biochemical parameters related to red blood cell production as well as markers of aerobic and anaerobic exercise performance. Cobalt is a required trace element in animals, but administration in excess is considered dangerous and potentially performance enhancing in equine athletes. In addition, for adequate regulation of naturally occurring substances, such as cobalt, its distribution among the population must be defined. In order to identify this distribution, plasma Cobalt was determined from 245 Standardbred horses with no cobalt supplementation from farms in New York and New Jersey, including horses at the Rutgers University Equine Science Center. Samples were analysed by Inductively Coupled Plasma Mass Spectrometry. Seven healthy, race fit Standardbreds (4 geldings, 3 mares, age: 5±3 years, ~500 kg) were used for the PED experiment. An incremental graded exercise test (GXT) to measure maximal aerobic capacity (V?O2max) and markers of performance, measurement of plasma volume and blood volume as well as the measurement of lactate, erythropoietin (EPO), and various blood haematological factors were determined 7 days prior to cobalt administration. Each horse was administered a sterile solution of cobalt salts (50 mg of elemental Co as CoCl2 in 10 ml of saline, IV) at 9 AM on three consecutive days via the jugular vein. Blood samples were obtained from the contralateral jugular vein before and at 1, 2, 4 and 24 h after administration. Plasma and blood volume were measured one day after the last dose of cobalt, and a post administration GXT was performed the next day. Horses were observed for signs of adverse effects of the cobalt administration (agitation, sweating, increased respiration, etc.). Plasma cobalt concentration increased from a pre-administration mean of 1.6±0.6 to 369±28 μg/l following 3 doses of the cobalt solution (P<0.05). This Co concentration was unaccompanied by changes in aerobic or anaerobic performance, plasma EPO concentration, plasma volume, resting blood volume, total blood volume, or estimated red blood cell volume (P>0.05). There were no observed adverse effects.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: McKeever, KH, HC Manso, Filho, Ellen Rankins, Camille Duchamp, Yassin Salah, CK Fenger, W Duer, K Malinowski, GA Maylin. Clenbuterol plasma concentrations after therapeutic administration in fit Standardbred Horses: Threshold recommendations. Comparative Exercise Physiology (In Press), 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: McKeever, K.H., Malinowski, K., Fenger, C., Duer, W., and Maylin, G.A., Evaluation of cobalt as a performance enhancing drug (PED). Comparative Exercise Physiology (https://doi.org/10.3920/CEP200001), 16:243252, 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Klein, D.J., K.H. McKeever, E. T. Mirek, T. Anthony. Metabolomic investigation of equine skeletal muscle in response to acute fatiguing exercise and chronic exercise training. Frontiers in Physiology 11: Article 10:1-15, 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Wallace, S., Rankins, E. M., and KH.. McKeever. Effects of Lactanase� on the plasma lactate response to an incremental exercise test in the horse. Comparative Exercise Physiology, https://doi.org/10.3920/CEP200007, 16: 243-259, 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Manso, H.C., H.E. Manso, M. Watford, K.H. McKeever. Abundance of the skeletal muscle Glut-4 glucose transport protein in Standardbred foals during development and exercise. Comparative Exercise Physiology, 16 (5)- Pages: 395 - 402. https://doi.org/10.3920/CEP200008, 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Klein, D.J., T. Anthony, K.H. McKeever. Changes in maximal aerobic capacity, body composition, and athletic capacity with prolonged training and detraining in Standardbred horses. Comparative Exercise Physiology 16: 187195, 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Klein, D.J., T. Anthony, K.H. McKeever. Metabolomics in equine sport and exercise. Journal of Animal Physiology and Animal Nutrition 2020; 00:1-9, https://doi.org/10.1111/jpn.13384 , 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Yigit, T., F. Han, E Rankins, J. Yi, K. McKeever, K. Malinowski Wearable IMU-based Early Limb Lameness Detection for Horses using Multi-Layer Classifiers , IEEE 16th International Conference on Automation Science and Engineering (CASE), Paper 185, pp.1-8, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Rankins, E.M., H.C. Manso, K. Malinowski, and K.H. McKeever. Muscular tension as a marker of stress in horses. muscle. American Society of Animal Science ASAS-CAS-WSASAS Joint Annual Meeting, J. Anim. Sci. 98, E-Suppl. 1, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Klein, D.J., T. Anthony, K.H. McKeever Acute Fatiguing Exercise Alters Sarco-endoplasmic Reticulum Stress-related Signaling in Equine Skeletal Muscle, The FASEB Journal, 33: Supplement 1, 2020.

Progress 04/01/19 to 09/30/19

Outputs
Target Audience:The target audience includes stakeholders in the horse industry including, owners, veterinarians, regulatory officials, legislators, and anyone interested in the horse industry and the care of the horse. The audience also includes the interested in basic and applied mamalian physiology and pharmacology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has allowed us to train multiple undergraduates conducting GH Cook Senior Thesis research. We are training one PhD student and one MS student. Furthermore, we have two visiting professors from Brazil conducting research in the laboratory. How have the results been disseminated to communities of interest?The results are being diseminated through scientific presentations made at national and international meetings, local, regional and national forums for horse owners; through the Equine Science Center portal and web page, and through Dr. McKeever's service on the Association of Racing Commisioners International's top scientific committee as well as the Harness Racing Medication Collaborative. What do you plan to do during the next reporting period to accomplish the goals?We will continue to conduct experiments in progress and will submit aproximately six journal articles to refereed journals.

Impacts
What was accomplished under these goals? Objective 1: We have presented talks and prepared manuscripts regarding our research on exogenously administered cobalt affacts the health of race horses and we have prepared a manuscript regarding the threshold to detect this performance enhancing practice. The paper also contains data on the effects of the drug on performance. We have also completed the analysis and are writing up papers associated with a four year project focused on a Type 5 PDE drug that has the potential to replace the controversial drug lasix. Studies currently underway include one that is looking at the effects of short term clenbuterol administration on bodycomposition. This study is also examining the pharmacokinetics associated with a three day administration followed by a 96 hour witholding period. The study addresses a major concern for regulators of the Harness racing industry and results will have a major impact on the policy of that part of the industry. Objective 2: We have completed an experiment that looked at the effects of the supplement Lactanase. This supplement is costly for horse trainers and owners and our results demonstrate that it does not have any effect as touted on the label. Objective 4: We have preparedfour papers on our metabolomic studies andare in the process of submitting them to refereed journals. Secondly, we are currently conducting a large project to examine the effects of lasix on the microbiome. Lasix is a diuretic that affects fluid balance and alterations in gut fluid balance can upset the microbiome. This collaborative project involves investigators from Brazil, University of Delaware, University of Kentucky, and Rutgers. Finally, a PhD student in the lab has been conducting studies of motion analysis and electromyographs to validate techniques that will be applied to examine the efficacy of Equine Assisted Activities on PTSD in Veterans.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Evaluation of cobalt as a performance enhancing drug in fit Standardbred race horses, 10th International Conference on Equine Exercise Physiology, Lorne, Australia, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Effects of a type-5 phosphodiesterase inhibitor on pulmonary artery pressure in race fit horses, 10th International Conference on Equine Exercise Physiology, Lorne, Australia, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Papel do Laborat�rio de Fisiologia do Exerc�cio Equino e do Equine Science Center no desenvolvimento do cavalos atletas, Universidade Federal Rural de Pernambuco, Departamento de Zootecnia, N�cleo de Pesquisa Equina, Recife, Brazil, 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Associa��o Brasileira dos M�dicos Veterin�rios de Equ�deos, Testando a efic�cia dos suplementos alimentares. Suplementos de alimenta��o para manter o desempenho e a sa�de. Funciona?, Sao Paulo, Brazil, 2019
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Associa��o Brasileira dos M�dicos Veterin�rios de Equ�deos, Idade e desuso em atletas: efeitos de destreinamento, ortografia, les�o e envelhecimento. Envelhecimento e como isso afeta a resposta ao exerc�cio no cavalo, Sao Paulo, Brazil, 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Associa��o Brasileira dos M�dicos Veterin�rios de Equ�deos, Novo medicamento para tratar Hemorragia pulmonar induzida por exerc�cio (informa��o de nosso estudo de 4 anos do EIPHISOL), Sao Paulo, Brazil, 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Associa��o Brasileira dos M�dicos Veterin�rios de Equ�deos, Fluidos e eletr�litos corporais: respostas ao exerc�cio e treinamento, Sao Paulo, Brazil, 2019.
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Klein, D.J., T. Anthony, K.H. McKeever. Impacts of acute exercise, chronic training and chronological age on sarco-endoplasmic reticulum stress and the unfolded protein response in mammalian skeletal muscle. Comparative Exercise Physiology