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Topic:Hemoglobin

Hemoglobin is an iron-containing protein found in the red blood cells of horses, responsible for the transport of oxygen from the lungs to the tissues and the return of carbon dioxide from the tissues to the lungs. It plays a vital role in maintaining cellular respiration and energy metabolism. The structure and function of hemoglobin can be influenced by various factors, including genetics, age, and health status. Research into equine hemoglobin includes studies on its biochemical properties, the impact of different physiological and pathological conditions on its levels, and its importance in performance and endurance. This page compiles peer-reviewed research studies and scholarly articles that explore the characteristics, regulation, and clinical implications of hemoglobin in equine physiology and health.
Effect of furosemide and furosemide-carbazochrome combination on exercise-induced pulmonary hemorrhage in Standardbred racehorses.
The Canadian veterinary journal = La revue veterinaire canadienne    November 3, 2009   Volume 50, Issue 8 821-827 
Perez-Moreno CI, Couëtil LL, Pratt SM, Ochoa-Acuña HG, Raskin RE, Russell MA.The objective was to quantify the effect of furosemide and carbazochrome on exercise-induced pulmonary hemorrhage (EIPH) in Standardbred horses using red blood cell count and hemoglobin concentration in bronchoalveolar lavage (BAL) fluid. Six healthy Standardbred horses with prior evidence of EIPH performed a standardized treadmill test 4 h after administration of placebo, furosemide, or furosemide-carbazochrome combination. Red blood cell (RBC) counts and hemoglobin concentrations were determined on the BAL fluid. The RBC count in BAL ranges were (2903-26,025 cells/microL), (45-24,060 cells/m...
Stability and reproducibility of ADVIA 120-measured red blood cell and platelet parameters in dogs, cats, and horses, and the use of reticulocyte haemoglobin content (CH(R)) in the diagnosis of iron deficiency.
Tijdschrift voor diergeneeskunde    May 13, 2009   Volume 134, Issue 7 272-278 
Prins M, van Leeuwen MW, Teske E.Modern laser-based haematology analysers such as the ADVIA 120 have species-specific software and offer the possibility of assessing new haematological parameters. These parameters have yet to be evaluated, and as these analysers are often used in referral laboratories, it is important to know whether the values of haematological parameters change during sample transport. Therefore, samples of EDTA-anticoagulated blood from nine healthy dogs and EDTA- and citrate-anticoagulated blood from six healthy horses were collected and stored at room temperature for 72 and 48 hours, respectively. In can...
[Free hemoglobin and tumor necrosis factor-alpha in the blood of horses with colic or acute colitis].
Berliner und Munchener tierarztliche Wochenschrift    December 18, 2008   Volume 121, Issue 11-12 440-445 
Kyaw WO, Uhlig A, Köller G, Sack U, Schusser GF.A total of 50 adult horses were classified into 4 groups: healthy horses (group 1, n = 14), horses with non-strangulation obstruction (group 2, n = 13), horses with strangulation obstruction (group 3, n = 11) and horses with acute colitis and severe inflammation (peritonitis or thrombophlebitis) (group 4, n = 12). Eleven euthanized horses recreated from group 3 (4 horses with small intestinal strangulation obstruction and hemolytic peritoneal fluid) and group 4 (7 horses with septic peritonitis) were taken as group 5. Free hemoglobin (fr. Hb) and tumor necrosis factor-alpha (TNFa) were measure...
Evaluation of three methods for measurement of hemoglobin and calculated hemoglobin parameters with the ADVIA 2120 and ADVIA 120 in dogs, cats, and horses.
Veterinary clinical pathology    June 7, 2008   Volume 37, Issue 2 173-179 doi: 10.1111/j.1939-165X.2008.00039.x
Bauer N, Moritz A.Besides flow cytometric detection of cellular hemoglobin (HGB) concentration, the ADVIA 2120 uses a novel cyanide-free colorimetric method to determine extracellular total HGB concentration. In human samples, the results are equivalent to those of the cyanmethemoglobin method on the ADVIA 120. Cyanide-free HGB measurement has not been evaluated in animal samples. Objective: The aim of this prospective study was to compare the 3 methods of HGB analysis on the ADVIA 2120 and ADVIA 120 in blood samples from dogs, cats, and horses. Methods: Consecutive fresh K(3)EDTA blood samples from 119 dogs, 1...
Plasmid growth hormone releasing hormone therapy in healthy and laminitis-afflicted horses-evaluation and pilot study.
The journal of gene medicine    February 28, 2008   Volume 10, Issue 5 564-574 doi: 10.1002/jgm.1170
Brown PA, Bodles-Brakhop A, Draghia-Akli R.In vivo electroporation dramatically improves the potency of plasmid-mediated therapies, including in large animal models. Laminitis and arthritis are common and debilitating diseases in the horse, as well as humans. Methods: The effects of growth hormone releasing hormone (GHRH) on healthy horses and on horses with laminitis that were followed for 6 months after a single intramuscular injection and electroporation of 2.5 mg of an optimized myogenic GHRH-expressing plasmid were examined. Results: In the first study on six healthy horses, we observed a significant increase in body mass by day 1...
Clinical and clinicopathologic variables in adult horses receiving blood transfusions: 31 cases (1999-2005).
Journal of the American Veterinary Medical Association    July 17, 2007   Volume 231, Issue 2 267-274 doi: 10.2460/javma.231.2.267
Hurcombe SD, Mudge MC, Hinchcliff KW.To determine clinical and clinicopathologic abnormalities in horses administered a blood transfusion and evaluate effects of blood transfusion on these variables. Methods: Retrospective case series. Methods: 31 adult horses that received > or = 1 blood transfusion. Procedures-Medical records of horses receiving a blood transfusion were reviewed to obtain clinical findings, laboratory test results before and after transfusion, adjunctive treatments, transfusion type and volume, response to transfusion, results of donor-recipient compatibility testing, adverse reactions, and outcome. Results:...
Clinical evaluation of the CA530-VET hematology analyzer for use in veterinary practice.
Veterinary clinical pathology    May 25, 2007   Volume 36, Issue 2 155-166 doi: 10.1111/j.1939-165x.2007.tb00202.x
Roleff S, Arndt G, Bottema B, Junker L, Grabner A, Kohn B.The CA530-VET is a completely automated impedance cell hematology analyzer, which yields a 16-parameter blood count including a 3-part leukocyte differential. Objective: The aim of this study was to examine the operational potential of the CA530-VET and its value for use in veterinary practice. Methods: The analyzer was tested for blood carry-over, precision, and accuracy. Comparison methods included the CELL-DYN 3500, microhematocrit centrifugation, manual platelet (PLT) counting for feline and equine species, and a 100-cell manual WBC differential. Blood samples for comparison of the methods...
Low dose exogenous erythropoietin elicits an ergogenic effect in standardbred horses.
Equine veterinary journal. Supplement    April 4, 2007   Issue 36 233-238 doi: 10.1111/j.2042-3306.2006.tb05545.x
McKeever KH, Agans JM, Geiser S, Lorimer PJ, Maylin GA.Recombinant human erythropoietin (rhuEPO) causes an increase in red blood cell production and aerobic capacity in other species; however, data are lacking on effects in the horse. Objective: This study tested the hypothesis that rhuEPO administration would alter red cell volume (RCV), aerobic capacity (VO2max) and indices of anaerobic power. Methods: Eight healthy, unfit mares accustomed to the laboratory and experimental protocols were randomly assigned to either a control (CON, n = 4; 3 ml saline 3 times/week for 3 weeks) or EPO group (EPO, n = 4, 50 iu/kg bwt rhuEPO/3 ml saline 3 times/week...
Nitric oxide synthase inhibition in thoroughbred horses augments O2 extraction at rest and submaximal exercise, but not during short-term maximal exercise.
Equine veterinary journal. Supplement    April 4, 2007   Issue 36 481-486 doi: 10.1111/j.2042-3306.2006.tb05591.x
Manohar M, Goetz TE, Hassan AS.Work is required to establish the role of endogenous nitric oxide (NO) in metabolism of resting and exercising horses. Objective: To examine the effects of NO synthase inhibition on O2 extraction and anaerobic metabolism at rest, and during submaximal and maximal exertion. Methods: Placebo and NO synthase inhibition (with Nomega-nitro-L-arginine methyl ester [L-NAME] administered at 20 mg/kg bwt i.v.) studies were performed in random order, 7 days apart on 7 healthy, exercise-trained Thoroughbred horses at rest and during incremental exercise leading to 120 sec of maximal exertion at 14 m/sec ...
The physiological responses to simulated race tests on a track and on a treadmill in standardbred trotters.
Equine veterinary journal. Supplement    April 4, 2007   Issue 36 123-127 doi: 10.1111/j.2042-3306.2006.tb05527.x
Nostell K, Funkquist P, Nyman G, Essén-Gustavsson B, Connysson M, Muhonen S, Jansson A.It is unclear to what extent the physiological response to a standardised treadmill exercise test simulating racing conditions resembles the circulatory and metabolic response observed after a simulated race on a track. Objective: To compare the physiological responses of a standardised treadmill exercise test used to simulate racing conditions and a simulated race performed on a track on the same Standardbred trotting horses, all in racing condition. Methods: Six Standardbred trotters in racing condition performed a standardised inclined treadmill exercise test protocol simulating racing cond...
Investigation of blood oxidant/antioxidant markers in healthy competition horses of different breeds.
Equine veterinary journal. Supplement    April 4, 2007   Issue 36 239-244 doi: 10.1111/j.2042-3306.2006.tb05546.x
Kirschvink N, de Moffarts B, Farnir F, Pincemail J, Lekeux P.There is increasing evidence that the equine athlete is exposed to exercise-induced changes of its oxidant/antioxidant balance and antioxidant supplementation is frequently recommended. However, it is unknown whether there is a specific need for antioxidants according to performance, breed, gender or age. Objective: To assess whether breed-, gender- and age-related differences of blood oxidant/antioxidant markers occur in competition horses. Methods: Healthy horses (n = 493) underwent oxidant/ antioxidant blood marker determination. Vitamin E, lipophilic antioxidant capacity (ACL), ascorbic ac...
Red blood cell erythropoietin, not plasma erythropoietin, concentrations correlate with changes in hematological indices in horses receiving a single dose of recombinant human erythropoietin by subcutaneous injection.
Journal of veterinary pharmacology and therapeutics    March 14, 2007   Volume 30, Issue 2 175-178 doi: 10.1111/j.1365-2885.2007.00828.x
Singh AK, Gupta S, Barnes A, Carlson JM, Ayers JK.No abstract available
The effect of colic on oxygen extraction in horses.
Veterinary journal (London, England : 1997)    February 23, 2007   Volume 175, Issue 1 102-107 doi: 10.1016/j.tvjl.2006.12.010
Cambier C, Wierinckx M, Grulke S, Clerbaux T, Serteyn D, Detry B, Liardet MP, Frans A, Gustin P.Blood oxygen transport and oxygen extraction were assessed in horses with colic. A gravity score (GS) ranging from 1 to 3 was attributed to each colic case with healthy horses used as controls. Jugular venous and carotid arterial blood samples were collected and concentrations of 2,3-diphosphoglycerate, adenosine triphosphate, inorganic phosphate and chloride were determined. pH and partial pressures of carbon dioxide (PCO(2)), and oxygen (PO(2)) were also measured. Oxygen equilibrium curves (OEC) were constructed under standard conditions and oxygen extraction ratios calculated. Haemoglobin o...
Shifts in the haemoglobin-oxygen dissociation curve: can we manipulate P50 to good effect?
Veterinary journal (London, England : 1997)    February 22, 2007   Volume 175, Issue 1 12-13 doi: 10.1016/j.tvjl.2007.01.003
Dugdale A.No abstract available
Comparison of plasma biochemical parameters in Thoroughbred and Purebred Arabian horses during the same-intensity exercise.
Polish journal of veterinary sciences    January 6, 2007   Volume 9, Issue 4 233-238 
Kedzierski W, Bergero D.The objective of the present study was to compare changes of blood parameters induced by the same work and performed in the same conditions in two racehorse breeds, Thoroughbred and Purebred Arabian. The effect of moderate-intensity exercise was studied in 20 stallions--ten Thoroughbreds, aged 2-3 years and the same number of Purebred Arabians, 3-4 years old. All the horses were administrated the same effort test consisting in 1200 m gallop at a speed typical of the daily training sessions. Three jugular venous blood samples were collected for each horse: at rest, just after the end of the gal...
Studies on some paraclinical indices on intoxication in horses from freshly cut Jimson weed (Datura stramonium)-contaminated maize intended for ensiling.
Journal of the South African Veterinary Association    December 2, 2006   Volume 77, Issue 3 145-149 
Binev R, Valchev I, Nikolov J.Monitoring of changes in some blood laboratory parameters in 34 horses after ingesting freshly harvested maize that was to be used for ensiling, heavily contaminated with young Datura stramonium plants, is described. For a 7-day period the following parameters were monitored: haemoglobin content (HGB), red blood cell counts (RBC), white blood cell counts (WBC), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), differential white cell counts (DWC), erythrocyte sedimentation rate (ESR), protein fractions, aspa...
Red maple (Acer rubrum) leaf toxicosis in horses: a retrospective study of 32 cases.
Journal of veterinary internal medicine    October 27, 2006   Volume 20, Issue 5 1197-1201 doi: 10.1892/0891-6640(2006)20[1197:rmarlt]2.0.co;2
Alward A, Corriher CA, Barton MH, Sellon DC, Blikslager AT, Jones SL.Ingestion of wilted red maple leaves by horses can result in severe hemolytic anemia and methemoglobinemia. Little is known about what factors influence the outcome of red maple leaf toxicosis in horses. Objective: Our hypothesis was that physical examination findings, clinicopathologic variables or therapeutic modalities may predict outcome in horses with red maple leaf toxicity. Methods: Horses with red maple leaf toxicosis presented to referral hospitals in the southeast region of the United States. Methods: A multi-institutional retrospective study was designed to identify factors that pre...
Pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses.
Veterinary clinical pathology    June 20, 2006   Volume 35, Issue 2 144-156 doi: 10.1111/j.1939-165x.2006.tb00108.x
Harvey JW.Deficiencies of enzymes involved in erythrocyte metabolism can have significant effects on erythrocyte function and survival. Animals with pyruvate kinase (PK) or phosphofructokinase (PFK) deficiencies have shortened erythrocyte life spans and regenerative anemia. PK-deficient dogs (but not PK-deficient cats) develop progressive myelofibrosis and osteosclerosis of bone marrow and hemochromatosis and cirrhosis of the liver. PFK-deficient dogs have sporadic episodes of hyperventilation-induced intravascular hemolysis and hemoglobinuria. Cytochrome b5 reductase (Cb5R) deficiency in dogs and cats ...
Hyperhydration prior to a simulated second day of the 3-day moderate intensity equestrian competition does not cause arterial hypoxemia in Thoroughbred horses.
European journal of applied physiology    May 10, 2006   Volume 97, Issue 4 462-470 doi: 10.1007/s00421-006-0191-z
Tennent-Brown BS, Goetz TE, Manohar M, Hassan AS, Freeman DE, Bundy JS, Evans MR.Dehydration and the associated impairment of cardiovascular and thermoregulatory function comprise major veterinary problems in horses performing prolonged exercise, particularly under hot and humid conditions. For these reasons, there is considerable interest in using pre-exercise hyperhydration to help maintain blood volume in the face of the excessive fluid loss associated with sweat production during prolonged exertion. However, recently it was reported that pre-exercise hyperhydration causes arterial hypoxemia in horses performing moderate intensity exercise simulating the second day of a...
Contribution of cooperativity and the Bohr effect to efficient oxygen transport by hemoglobins from five mammalian species.
Zoological science    March 21, 2006   Volume 23, Issue 1 49-55 doi: 10.2108/zsj.23.49
Zhang Y, Kobayashi K, Kitazawa K, Imai K, Kobayashi M.By using published experimental values of the standard oxygen (O2) equilibrium curve and the in vivo arterial and venous O2 pressure (PO2) of fetal and maternal blood in five mammalian species (human, cow, pig, sheep, and horse), we investigated the relationship between the efficiency of O2 delivery and the effectiveness of the Bohr shift, and discussed the significance of cooperativity for mammalian Hb. The O2 delivery of fetal blood was more efficient than that of maternal blood, and the effectiveness of the Bohr shift at both O2 loading and release sites of fetal blood was high. A linear re...
Can a membrane oxygenator be a model for lung NO and CO transfer?
Journal of applied physiology (Bethesda, Md. : 1985)    January 5, 2006   Volume 100, Issue 5 1527-1538 doi: 10.1152/japplphysiol.00949.2005
Borland C, Dunningham H, Bottrill F, Vuylsteke A.To model lung nitric oxide (NO) and carbon monoxide (CO) uptake, a membrane oxygenator circuit was primed with horse blood flowing at 2.5 l/min. Its gas channel was ventilated with 5 parts/million NO, 0.02% CO, and 22% O2 at 5 l/min. NO diffusing capacity (Dno) and CO diffusing capacity (Dco) were calculated from inlet and outlet gas concentrations and flow rates: Dno = 13.45 ml.min(-1).Torr(-1) (SD 5.84) and Dco = 1.22 ml.min(-1).Torr(-1) (SD 0.3). Dno and Dco increased (P = 0.002) with blood volume/surface area. 1/Dno (P < 0.001) and 1/Dco (P < 0.001) increased with 1/Hb. Dno (P = 0.01...
Neonatal isoerythrolysis in horse foals and a mule foal: 18 cases (1988-2003).
Journal of the American Veterinary Medical Association    November 4, 2005   Volume 227, Issue 8 1276-1283 doi: 10.2460/javma.2005.227.1276
Boyle AG, Magdesian KG, Ruby RE.To assess data regarding clinical features, clinicopathologic and blood gas variables, and outcome from horse and mule foals with confirmed neonatal isoerythrolysis (NI). Methods: Retrospective case series. Methods: 17 horse and 1 mule foals. Methods: Medical records of foals (< 14 days old) with NI were reviewed. Information collected included signalment; clinical examination findings; results of hematologic, serum and plasma biochemical, and venous blood gas analyses and urinalysis; treatments; and outcome. Results: Data from 17 horse foals and 1 mule foal with NI (mean age, 71 hours) were e...
Transvascular fluid flux from the pulmonary vasculature at rest and during exercise in horses.
The Journal of physiology    November 3, 2005   Volume 570, Issue Pt 2 397-405 doi: 10.1113/jphysiol.2005.098723
Vengust M, Staempfli H, Viel L, Heigenhauser G.Exercise causes changes in pulmonary haemodynamics through redistribution of blood flow, increase in the pulmonary surface area, and increase in pulmonary vascular pressures. These changes contribute to the increase in fluid exchange across the alveolar-capillary barrier. To determine the extent of the fluid exchange across the alveolar-capillary barrier at rest and during exercise, six horses were exercised on a high-speed treadmill until fatigue. Arterial and mixed venous blood were sampled at rest and during exercise and recovery. Blood volume changes across the lung (DeltaBV; measured in p...
Reticulocyte changes after experimental anemia and erythropoietin treatment of horses.
Journal of applied physiology (Bethesda, Md. : 1985)    August 17, 2005   Volume 99, Issue 3 915-921 doi: 10.1152/japplphysiol.00438.2005
Cooper C, Sears W, Bienzle D.Availability of recombinant human erythropoietin (EPO) has facilitated use to enhance red blood cell production, and therefore aerobic performance, in human and equine athletes. Recombinant human EPO promotes growth and differentiation of equine erythroid precursor cells, but in some horses repeat administration induces immune interference with endogenous EPO resulting in fatal anemia. Although blood reticulocyte parameters acquire unique changes in humans treated with EPO, with manual enumeration methods, horses were not considered to release reticulocytes from the bone marrow into circulatio...
The pharmacokinetics of hemoglobin-based oxygen carrier hemoglobin glutamer-200 bovine in the horse.
Anesthesia and analgesia    May 28, 2005   Volume 100, Issue 6 1570-1575 doi: 10.1213/01.ANE.0000154081.38466.09
Soma LR, Uboh CE, Guan F, Luo Y, Moate PJ, Boston RC, Driessen B.Hemoglobin-glutamer-200 (HBOC-200) is a hemoglobin (Hb)-based oxygen carrier (HBOC) comprising glutaraldehyde-polymerized bovine Hb. In this study, we sought to determine the pharmacokinetics of this first generation HBOC after IV infusion of 32.5 g of HBOC-200 solution in horses. Quantification of HBOC-200 in equine plasma and urine was performed using a method recently developed by our laboratory. The elimination from plasma was based on size distribution of the bovine Hb polymer. The decline of plasma concentration-time curve of HBOC-200 was described by a noninterchanging 2-compartmental m...
A new relaxed state in horse methemoglobin characterized by crystallographic studies.
Proteins    May 12, 2005   Volume 60, Issue 3 547-551 doi: 10.1002/prot.20510
Sankaranarayanan R, Biswal BK, Vijayan M.A new relaxed state has been characterized in the crystals of horse methemoglobin grown at neutral pH at low ionic concentration and their low humidity variants. The crystals provide an example for improvement in X-ray diffraction quality with reduced solvent content. Only the classical R state has been so far observed in liganded horse hemoglobin. The state characterized in the present study lies in between the R state and the R2 state characterized earlier in liganded human hemoglobin. The results presented here, along with those of earlier studies, suggest that relaxed and tense hemoglobin ...
Blood-oxygen binding in healthy Standardbred horses.
Veterinary journal (London, England : 1997)    February 25, 2005   Volume 169, Issue 2 251-256 doi: 10.1016/j.tvjl.2004.01.025
Cambier C, Di Passio N, Clerbaux T, Amory H, Marville V, Detry B, Frans A, Gustin P.The purpose of this study was to determine the effect of regulating factors on the oxygen equilibrium curve (OEC) under standard conditions and then to calculate the oxygen extraction between arterial and jugular venous blood in healthy Standardbred horses. The results were compared to those previously obtained in humans and cattle, using the same experimental method. The partial oxygen pressure at 50% saturation of haemoglobin, measured under standard conditions (standard P50), was 24.8+/-2.0 (SD of mean) mmHg. This value was similar to the cattle standard P50 (25.0+/-1.4 mmHg, SD of mean) bu...
Measurement of erythrocyte carbonic anhydrase isozymes (CA-I and CA-II) in racehorses and riding horses.
The Journal of veterinary medical science    February 9, 2005   Volume 67, Issue 1 63-67 doi: 10.1292/jvms.67.63
Nishita T, Takahasi M, Kasuya T, Matsui K, Ichihara N, Murakami M, Asari M.Equine carbonic anhydrase isozymes (CA-I and CA-II) were purified from erythrocytes by several column chromatography. Polyclonal anti-CA-I and anti-CA-II sera were produced in rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISA) were established to determine the developmental changes in CA-I and CA-II levels in equine erythrocytes. Concentrations of CA-I and CA-II in erythrocytes from 150 clinically normal thoroughbreds (123 racehorses and 27 riding horses) were determined by ELISA. Mean (+/- SD) concentrations of CA-I and CA-II in racehorses were 1.70 +/- 0.48 and 0.94 +/...
The effects of automated plasmapheresis on clinical, haematological, biochemical and coagulation variables in horses.
Veterinary journal (London, England : 1997)    February 3, 2005   Volume 169, Issue 1 102-107 doi: 10.1016/j.tvjl.2004.01.007
Feige K, Ehrat FB, Kästner SB, Wampfler B.The goal of this study was to determine the effects of plasmapheresis on the behaviour, general condition, haematological, biochemical and coagulation variables of donor horses for 32 days following the procedure. Twenty millilitres of plasma/kg body weight were collected via plasmapheresis in six clinically healthy horses. The general behaviour and condition of the horses was not affected by the procedure. During plasmapheresis, there was a mild increase in the haematocrit, haemoglobin concentration and total erythrocyte and leucocyte counts (P < 0.01). The mean concentrations of total pro...
Whole blood, plasma viscosity, and erythrocyte aggregation as a determining factor of competitiveness in standard bred trotters.
Clinical hemorheology and microcirculation    January 25, 2005   Volume 32, Issue 1 31-41 
Stoiber B, Zach C, Izay B, Windberger U.In situations where capillary perfusion in skeletal muscles is limited, changes in blood flow play an important role. Especially alterations in intrinsic erythrocyte factors like red cell aggregation and deformability would increase blood flow resistance. In our study we investigated whether whole blood and plasma viscosity influence exercise tolerance during submaximal training and whether a difference can be realized between trained and untrained standardbred trotters. Venous blood from 42 healthy adult trotters (20 horses at the beginning of their training and 22 well trained horses) was in...
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