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THE EFECTS OF COMBINED RIDER AND TACK WEIGHT ON THE LACTIC ACID PRODUCTION IN THE HORSE ( Equus caballus) AT THREE DIFFERENT GAITS: WALK, TROT, AND CANTER. Anahita A. Ariarad and Allison S. Lindsay Department of Biological Science Saddleback College Mission Viejo, CA 92692 During glycolysis, L-Lactate is produced from pyruvate via the enzyme lactate dehydrogenase (LDH) during normal metabolism and exercise. Due to increased recruitment of skeletal muscle during exercise, it was predicted that lactate levels in the horses would increase when the combined weight of the rider and tack was added. To test this, blood lactate levels of five horses were taken before exercise and again after walking, trotting, and cantering for various lengths of time. There was found to be a 37.5% increase/decrease difference (say increase/decrease OR difference, this is redundant) between the values of the canter when comparing unmounted versus mounted blood lactate levels (p = 0.026, Tukey Correction). This data partially supports our hypothesis, but further investigation is needed. Introduction Lactic acid is produced in minute amounts during rest but in greater quantities during intense exercise. It is naturally present in humans as well as animals. When the oxygen level in the body is normal, carbohydrate^s breaks down into water and carbon dioxide. When the oxygen level is low, carbohydrate^s breaks down for energy and makes lactic acid. Lactic acid is formed from glycogen by muscle cells when the oxygen supply is inadequate to support energy production (Wickler and Gleeson, 1993). Buildup of lactic acid in the muscle occurs only in (during might be a better word here, rather than in) short bouts of exercise due to high intensity and it is usually correlated to fatigue, exhaustion and muscle soreness. During aerobic exercise, the heart and lungs supply adequate amounts of oxygen to the body for energy. Anaerobic exercise forces the body to demand more oxygen than the lungs and heart can supply. This shows that the energy supply is less and thus causes a high lactic acid level in the blood. (This sentence doesn’t really make sense) Typically anaerobic exercise forces a person to slow down because lactic acid build up causes moderate to severe muscle throbbing and rigidity. In human athletes, ATP production in (lower-case g) Glycolysis may be as high as 3mMol/g body weight x s (Newsholme & Leech^, 1983), and although similar estimations from equine muscle are unavailable, the comparison of the activities of key glycolytic enzymes in equine muscles to those in human muscles (Essen-Gustavsson et. al. 1984, Henriksson et. al. 1986, Roneus et. al. 1991, Cutmore et. al. 1993) indicates that anaerobic ATP production may be equally important in horses. Lactic acid gathers in the muscles when the supply of oxygen is scarce for the oxidative processes and quickly diffuses out into the blood stream. As lactic acid diffuses out of the muscles and other tissues, it appears in the blood as lactate. Blood lactate can be useful for evaluation ^of performance. Lactate, which is produced by the body all day long, is re-synthesized by the liver from glucose that provides the body with energy. In most mammals, lactate formed during exercise is oxidized to carbon dioxide and water (Wickler and Gleeson^, 1993). Under extreme conditions, horses contracting muscles are fueled by aerobic and anaerobic metabolic processes. When a horse performs or exercises, they use their muscles to accomplish tasks. (You probably don’t need to say this, as it is obvious) As lactic acid is produced in the muscles it leaks out into the blood and is then carried around the body. (You already said this) If this condition continues, the functioning of the body can then become impaired and the muscles can fatigue very rapidly. When oxygen becomes available, the lactic acid is converted to pyruvic acid and then into carbon dioxide, water and ATP (Kobayashi^, 2007). Horses performing low intensity exercise for long periods lose large amounts of sweat. Lower intensity exercise uses oxygen to provide energy, and is known as aerobic exercise. (You might want to give this definition earlier in the paper, before you discuss the effects of aerobic exercise) Aerobic exercise does not produce high levels of lactic acid. Muscles in horses secrete Dlactate as a by product (one word) of energy production during anaerobic exercise. In this study, the effects of exercise and the cause it has on the increase of ^on blood lactate levels in horses was tested. The objective was to determine if lactic acid levels in the horse (Equus caballus) increase^d between mounted and unmounted at each gait. It was expected that there would be a difference amid the blood lactate production between mounted and unmounted (unmounted is not actually a word) after all three gaits. Materials and Methods Five horses were used in this study to determine if the combined weight of the rider and tack has a significant affect sp. on blood lactate levels after walk, trot, and canter. All testing took place at the J.F. Shea Center for Therapeutic Riding in San Juan Capistrano, California under the supervision of a board-certified vet, Dr. Richard Markel. The horses were chosen for testing based on size, temperament, and soundness. This information was available in their medical/ health records and known by staff. Horses were removed from their stalls one at a time and a fresh, sterile needle was inserted into the jugular vein. The first two drops of blood were discarded before the base level sample was taken and read by the Lactate Scout (Sports Resource Group, Inc.). Over a period of a week, all five horses were lunged (define this word) on a line by a staff member in a twenty meter diameter round pen. Each horse walked and trotted for fifteen minutes at each gait, and cantered for six minutes. (Define walking, trotting and cantering.) Blood was taken in the same manner as before and analyzed by the Lactate Scout immediately after each gait. From 8 November to 10 November 2009, all horses were ridden in the same tack (define tack) and by the same rider to reduce variability in weight. Horses were ridden for the same amount of time (what amount of time?) at the walk, trot, and canter as in the unmounted tests. Blood was taken and analyzed in the same manner as before. All horses were then weighed using Blue Seal Horse and Pony Height and Weight Tape (Blue Seal Feeds Inc., Lawrence, Massachusetts). All data were transferred to MS Excel (Microsoft Corporation, Redmond, Washington) where data were analyzed by ANOVA and Tukey Correction (if p ≤ 0.05). Results The mean unmounted blood lactate level at the walk was 0.0005 ± 1.13 x 10-04 mM•L1 •kg-1 (±SEM, N=5), at the trot was 0.0004 ± 8.9941 x10-05 mM•L-1•kg-1 (±SEM, N=5), and at canter was 0.0008 ± 9.853 x 10-05 mM•L-1•kg-1 (±SEM, N=5). The mounted blood lactate level at the walk was 0.0003 ± 4.17 x 10-05 mM•L-1•kg-1 (±SEM, N=5), at the trot was 0.0003 ± 2.1 x1005 mM•L-1•kg-1 (±SEM, N=5), and at the canter was 0.0005 ± 8.25 x 10-05 mM•L-1•kg-1 (±SEM, N=5). As seen in Figure 1, between the groups, no difference was found when comparing mounted and unmounted values in the walk and trot (p = 0.164 and p = 0.348, respectively). In the canter group a ^significant difference was found (p = 0.026). Within the groups, a difference was found between the unmounted values for trot and canter (p = 0.007), and the values for walk and canter (p = 0.044). Similarly there was a difference found between the mounted values for trot and canter (p = 0.006) and walk and canter (p = 0.028). This section should be justified. 0.001 0.0009 mM • L-1 • kg-1 0.0008 0.0007 0.0006 Unmounted 0.0005 Mounted 0.0004 0.0003 0.0002 0.0001 0 Walk Trot Canter Figure 1. Mean combined lactate levels at walk, trot and canter unmounted versus mounted. No difference was found between unmounted and mounted blood lactate levels in the walk and trot groups. A difference was found between unmounted and mounted blood lactate levels in the canter group (p = 0.026, Tukey Correction). Discussion Lactic acid is capable of releasing energy to re-synthesize adenosine triphosphate (ATP) without the involvement of oxygen. Lactic acid is produced from pyruvate in the glycolysis cycle via the enzyme lactate dehydrogenase (LDH) during normal metabolism and exercise. The amount of lactate present after exercise can be a helpful tool in determining performance because it is an estimation of aerobic capacity (Poso, 2002). Within the unmounted group, no difference was found between the walk and the trot values. However, the trot to canter and walk to canter comparisons showed a significant difference in blood lactate values. Similarly within the mounted group the walk to trot comparison revealed no difference, where the trot to canter and walk to canter assessments discovered a significant difference. The hypothesis for this project stated that blood lactate levels would be higher at all three gaits while mounted versus unmounted. However, collected data showed that blood lactate levels were higher in the unmounted group. No statistical difference was found when comparing the mounted and unmounted levels in both the walk and the trot. This is most likely because the animals were not pushed into a state of anaerobic respiration at these gaits. For this same reason, there was a difference found in the canter values between the groups. Though there are several variables that can be taken into account when examining blood lactate levels, the researchers believe that the results in this experiment could be explained by looking at the level of energy applied when comparing exercise by lunging versus riding. During the unmounted testing, horses showed a higher energy exertion at the trot and canter when compared to the mounted testing. This is evidenced by the amount of forward momentum at each gait while lunging versus under saddle. Though the researchers did not measure the velocity of each animal was not measured (wasn’t in passive voice), obvious differences were observed while testing. Though it is possible to push a horse into a higher energy level while mounted, our rider did not attempt to do this. Though this experiment did find a significant difference, the relationship between blood lactate production and increase in load (?) was opposite to what was originally hypothesized. In a study to determine lactate minimum speed (LMS), the individual lactate production and removal rates, in horses, Gondim et al. (2007) found no difference in blood lactate concentration at rest and at LMS, despite an increase in heart rate. The data found in both these studies is inconsistent with the majority of information available on blood lactate. LMS has previously been tested in basketball players and runners (Tegtbur et al., 1993), in swimmers (Ribeiro et al., 2003) and rats (Voltarelli et al., 2002) as well. In all the above studies lactate levels at LMS were significantly higher than those at rest (Gondim et al., 2007). All of the above experiments indicate that there exist one or more key differences in the processing of post-exercise lactate in humans and equines. There are several factors that can affect the lactate concentration in blood and these ^ factors need to be accounted for when blood lactate measurement serves as a marker of performance. The rate of lactate production in exercising muscle is influenced by oxidative capacity and thus training, which is often accompanied with an increase in the number of mitochondria, may reduce lactate production (Poso, 2002). On top of this, horses already have a marked increase in oxygen consumption with a maximal oxygen uptake of about 160 ml/kg body weight × min (Evans & Rose 1988, Rose et al. 1988). This is more than twice the uptake in human elite athletes (Poso, 2002). Training can also increase the monocarboxylate transport proteins in the sarcolemma (Poso, 2002, Hashimoto et al., 2008, Brooks et al., 1999). This allocates for a faster rate of facilitated diffusion and therefore would add to the lactate concentration. The quantity of lactic acid that is permitted to build up is determined by the effort that is needed to increase the lactate concentration to levels above its resting value. This occurs when anaerobic glycolysis produces lactate at a greater rate than the animal’s capacity to remove it (Gondim et al., 2007). In skeletal muscle, the fast-twitch glycolitic fibers are mainly producers of lactate while the slow oxidative fibers act as consumers; and therefore these two fibers, along with other factors, are responsible for creating the net change in lactate levels (Hashimoto et al., 2008). Acknowledgements We would like to thanks the J.F. Shea Therapeutic Riding Center for allowing us the use of their facilities, horses, medical supplies and time of their staff, with a special thanks to Richard Markel, DVM. We would also like to thanks the following people for their assistance and input to this project: Professor Steve Teh, Aaron Ko, and Amir Zand. Literature Cited Brooks, G.A., H. Dubouchaud (Dubouchand, H.), M. Brown (Brown, M.), J.P. Sicurello (Sicurello, J.P.), and ^& C.E. Butz (Butz, C.E.). 1999. Role of mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular lactate shuttle. Proceedings of the National Academy of Sciences of the United States of America 96: 1129-1134. Cutmore, C.M.M., D.H. Snow (Snow, D.H.), and E.A. Newsholme (Newsholme, E.A.). 1993. Activities of key enzymes of aerobic and anaerobic metabolism in middle gluteal muscle from trained and untrained horses. Equine vet. J. 17: 354-356. Essén-Gustavsson, B., K. Karlström ( Karlstrom, K.), and A. Lindholm (Lindholm, A.).1984. Fibre types, enzyme activities and substrate utilization in skeletal muscles of horses competing in endurance races. Equine vet. J. 16: 197-202. 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Chronic stimulation of mammalian muscle: changes in enzyme of six metabolic pathways. Am. J. Physiol. 251: C614-C632. Kobayashi, M. 2007. Simple Lactate Measurement in Horses Using a Portable Lactate Analyzer with Lancet Skin Punctures Under Field Conditions. Journal of Equine Science 18: 5-11. Newsholme, EA ^& A.R. Leech (Leech, A.R.). Biochemistry for the Medical Sciences. Chichester, John Wiley & Sons: 1986, pp 357-381. Poso, A.R. 2002. Monocarboxylate transporters and lactate metabolism in equine athletes: a review. Acta Veterinaria Scandinavica 43: 63-74. Ribeiro, L., P. Balikian (Balikian, P.), P. Malachias (Malachias, P.), and ^& V. Baldissera (Baldissera, V.). 2003. Stage length, spline function and lactate minimum swimming speed. J. Sports Med. Phys. Fitness 43: 312-318. Roneus, N., B. Essen-Gustavsson (Essen-Gustavsson, B.), A. Lindholm (Lindholm, A.), and ^& S. Persson (Persson, S.). 1999. Muscle Characteristics and Plasma Lactate and Ammonia Response After Racing in Standardbred Trotters: Relation to Performance. Equine Veterinary Journal 31 (2):170-173. Rose, R.J., D.R. Hodgson (Hodgson, D.R.), T.B. Kelso (Kelso, T.B.), L.J. McCutcheon (McCutchneon, L.J.), T.A. Reid (Reid, T.A.), W.M. Bayly (Bayly, W.M.), and ^& P.D. Gollnick (Gollnick, P.D.). 1988. Maximum O2 uptake, O2 debt and deficit, and muscle metabolites in Thoroughbred horses. J. appl. Physiol 64: 781-788. Tegtbur, U., M.W. Busse (Busse, M.W.), and ^& K.M. Braumann (Braumann, K.M.). 1993. Estimation of an individual equilibrium between lactate production and catabolism during exercise. Med. Sci. Sports Exerc. 25: 620–627. Voltarelli, F.A., C.A. Gobatto (Gobatto, C.A.), and ^& M.A. de Mello (de Mello, M.A.). 2002. Determination of anaerobic threshold in rats using the lactate minimum test. Braz. J. Med. Biol. Res. 35 (11): 1389–1394. Wickler, S.J. and ^& T.T. Gleeson (Gleeson, T.T.). 1993. Lactate and glucose metabolism in mouse (Mus musculus) and reptile (Anolis carolinensis) skeletal muscle. American Journal of Physiology – Regulatory, Integrative and Comparative Physiology 246: 487-491. Review Form Department of Biological Sciences Saddleback College, Mission Viejo, CA 92692 Author (s): Anahita A. Ariarad and Allison S. Lindsay_______________ Title: The Effects of Combined Rider and Tack Weight on the Lactic Acid Production in the horse (Equus caballus) at Three Different Gaits: Walk, Trot and Canter Summary Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper. In glycolysis, L-lactate is produced from pyruvate. It was predicted that with increased weight, and therefore increased effort, more lactate would be produced when a rider and saddle were on the horse during exercise. Buildup of lactic acid occurs during bursts of energy. It has been shown that this is important in humans, and so it can be assumed that it is important in horses. The point of this project was to determine if there was a difference in blood lactate levels in mounted verses unmounted horses. Five horses were used. There blood lactate levels were tested before and after testing. A significant difference between mounted and unmounted horse blood lactate levels was only found when the horses cantered. There might have shown no significance between the lactate levels in the trot and walk because there was not a big enough difference in the mounted verses unmounted weight, or the horses may not have been exercised long enough. General Comments Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of knowledge. The project idea is well thought out, and well executed. There seems to be some information in the introduction and discussion that is extraneous to the project. The paper overall seemed a bit wordy, but very thorough. The idea for the project is interesting. It’s relateableness to human physiology makes it interesting. I don’t see what the importance of it really would be. I don’t think that they were exploring a very new idea. Muscles get tired due to lactic-acid build-up. That is a known fact. It seems like a lot of the references were used just for the sake of having lots of references. Technical Criticism Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript. This paper was a final version This paper was a rough draft Unmounted is not a word (I looked it up just to be sure), but they use it about 50 times in their paper. They have a few sentances which do not make sense. The order in which they give information was good. They forgot commas in most of their in-text citations, and put first initials before the last names in all of their literature cited. They used and instead of & in their literature cited as well. Most of the paper is in passive voice, but not quite all of it. Most of the paper was justified, but not quite all of it. Correction Key: Delete ^insert Sp., spelling