Training Effects on the Stress Predictors for Young Lusitano Horses Used in Dressage.

The purpose of this study was to investigate stressful responses during a 6-week training protocol in young Lusitano horses used for dressage. The hypothesis was that the proposed training protocol would improve fitness and ensure the welfare of the animals by reducing stress predictors. Nine 4-year-old horses were evaluated before (M1) and six weeks after (M2) beginning a training protocol. The training program was performed six times per week and included 40−80 min of individually intensity-adjusted preparatory exercises for dressage. For both moments, the horses were examined before (T0) and after (T1) dressage simulation tests (DST), and at 30 (T2) and 240 min (T3) during the recovery period. Blood samples were taken to determine the horses' cortisol levels, total WBC, and neutrophil and lymphocyte counts. All variables were analyzed by one-way ANOVA and Tukey tests, with p ≤ 0.05. After training, there was a significant reduction in cortisol (p = 0.0133), HR (p = 0.0283), total WBC (p < 0.0001), and neutrophil (p < 0.0001) and lymphocyte (p = 0.0341) counts. Other findings included an increase in HRV parameters related to a cardiac vagal modulation. In conclusion, the chosen training protocol led to better fitness as the horses worked more intensively with lower cardiovascular requirements, and they showed blunted cortisol responses at M2. Such data can be used to evaluate performance, but also to predict the welfare of athletic horses.

Effect of Clenbuterol on Muscle Activity During Exercise in Standardbred Horses.

Clenbuterol (ß2 agonist) is a commonly administered bronchodilator in race and performance horses. While long-term administration can alter exercise performance and muscle properties, little is known about its effects on these parameters following short-term administration. A single dose of clenbuterol (0.80 µg/kg) was expected to alter muscle activity of the extensor carpi radialis, semitendinosus, and longissimus dorsi during submaximal exercise. Eight mature Standardbred horses exercised for 2 minutes at 5 m/s on a high-speed treadmill following clenbuterol dosing (clenbuterol) or no dosing (control) in a crossover experimental design. Surface electromyography (sEMG) data were collected continuously from the muscles of interest and processed to determine average rectified value (ARV) and median frequency (MF) of the signal during peak muscle activation (100 ms period) during 15 strides. ARV data were log transformed. Data were analyzed with a mixed model ANOVA with fixed effects of period and treatment and a random effect of horse. No differences (P > .05) in amplitude (ARV) or frequency (MF) of the EMG signal were detected following clenbuterol administration. Thus, a one-time dose of clenbuterol had no statistically detectable effect on muscle activity during submaximal exercise. Further studies should be undertaken to confirm these results and examine the effects of long-term administration on muscle activity during exercise.

Muscular tension as an indicator of acute stress in horses.

Horses' muscular tension during acute stress remains unexplored. Our aim was to assess muscular, behavioral, cortisol, and hematocrit responses to social isolation (ISO), novel object exposure (NOV), and sham clipping (CLIP). Altered stress responses were expected. Eight mature Standardbred horses (four mares and four geldings) were exposed to acute stressors and a control period (CON) in a balanced, replicated 4×4 Latin Square experimental design with 3 min treatment periods and 10 min washout periods. Surface electromyography collected from the masseter, brachiocephalas, cervical trapezius, and longissimus dorsi was processed to derive average rectified value (ARV) and median frequency (MF) during the initial, middle, and final 30 s of treatments. ARV and MF data were log transformed then analyzed using a mixed model, repeated measures ANOVA along with plasma cortisol and hematocrit. Behavior data were analyzed using a negative binomial distribution mixed model ANOVA. CLIP resulted in greater (p < 0.05) log ARV in the masseter (1.5 + 1.5%, mean + SD) and brachiocepahlas (2.2 + 2.0%) than CON (-1.2 + 1.4%, 0.1 + 1.5%). ISO resulted in greater (p < 0.05) log ARV in the masseter (0.2 + 1.3%) and cervical trapezius (0.6 + 1.3%) than CON (-1.2 + 1.4%, -1.0 + 1.7%). ISO increased (p < 0.05) the total number of stress-related behaviors and hematocrit. No changes in cortisol were observed. We suggest that muscular tension can be used as an indicator of acute stress in horses. Incorporating muscle activity into an array of measurements may provide a more nuanced understanding of stress responses.

How Much Energy Vaquejada Horses Spend in a Field Simulation Test?

Vaquejada is a high-intensity and short-duration exercise in which helper horses (HH) are responsible to keep a bull running in a line while pull horses (PH) work to put the bull down after 100 m of running. The purpose of this study was to quantify and compare energy expenditures (EE), transport costs (COT) and metabolic energy requirements (Pmet) of horses used in Vaquejada. Thus, eight Quarter Horses, in randomly formed pairs, performed a vaquejada simulation test (VST), which consisted of three races on a sand track (130-150 m), with a 5-min interval between them. All horses used an integrated heart rate (HR) and GPS monitoring system (V800, Polar Electro) and, from these data, EE, COT and Pmet were calculated using the formulas: EE (J/kg/min) = 0.0566 × HR1.9955, COT = (HR-35)/kg/m × 103 and Pmet = (HR-35)/min/kg. Blood samples were collected for lactate analysis at rest, immediately after the first, second and third race and after 30 min of recovery. Data obtained were submitted to one-way ANOVA and Tukey tests (p ≤ 0.05). In VST, HH had higher EE and higher HR at trot; while PH presented higher EE and HR at canter. Finally, considering total VST, PH had higher EE and COT, while HH had higher Pmet. Lactate was higher in PH. Despite practicing the same sport, PH and HH should be considered distinct athletes, and these must be considered to set up appropriate physical and nutritional programs, which will lead to better performance and guarantees of well-being.

Comparative Trial of Continuous Flow Enteral and Intravenous Fluid Therapy in Horses.

Continuous flow enteral fluid therapy with isotonic and hypotonic enteral electrolyte solutions are as safe and effective as intravenous fluid therapy. The aim of this study was to carry out a comparative assessment between continuous flow enteral and intravenous (IV) fluid therapy in adult experimentally dehydrated horses. Six experimentally dehydrated adult mares were used in a study carried out in a 6 × 3 crossover design, which each animal received three different treatments (isotonic enteral fluid therapy-EsISO, hypotonic enteral fluid therapy-EsHYPO and intravenous fluid therapy with Lactate Ringer Solution-LR IV, all in continuous flow). Solutions were administered at a rate of 15 mL-1.kg-1.h-1 for 8 h, after 36 h of water and food deprivation. Serum and urinary biochemical assessment; urinary volume, pH and specific gravity; and blood gas analysis were measured at -36, 0, 2, 4, 6, and 8 h. The dehydration period (DP) caused discrete hydroelectrolytic and acid base imbalances. The EsISO, EsHYPO and LR IV increased blood volume. Enteral solutions restored the imbalances yielded by the DP and all treatments increased urine volume. Also, the EsHYPO and LR IV showed no effects in acid base balance, while EsISO showed slightly acidifying effect. The present study certifies the efficacy and safety of isotonic and hypotonic continuous flow enteral fluid therapy in comparison to IV fluid therapy in dehydrated horses.

Is There an Ideal Rest Interval Between Races During Vaquejada in Which It Would Be Possible to Associate Best Performance and Welfare?

Vaquejada is an important Brazilian equine discipline. Understanding physiological adaptations of these athletes is crucial to improve properly performance, guaranteeing welfare. The objective of this study was to evaluate the influence of three vaquejada simulation tests (VST) on physiological parameters of horses and standardize a possible rest interval between races. Ten clinically healthy Quarter horses, 8.9 ± 4.3 year-old and 441.3 ± 25.0 kg, executed three VST, 5 days apart from each other. Vaquejada simulation tests consisted of two horses, a puller, and a helper, running with a bull on a soft sand track in which they must put the bull down. On M1, they ran three times with a 5-min rest between races; on M2, with a 10-min rest; and M3, with a 15-min rest. Clinical evaluation and blood sampling were made in all VST, before (T0), immediately after first run (T1), second run (T2), third run (T3) and at 30 minutes (T4), and 4 hours (T5) of recovery. Variables were statistically analyzed with a bifactorial comparison (P < .05). Exercise increased heart rate (HR), respiratory rate, body temperature (BT), lactate, triglycerides, packed cell volume, RBC, and hemoglobin concentration, with higher values in pull horses due to a more intense exercise. With 15-min of rest interval, helper horses showed lower values of glucose, aspartate aminotransferase, creatine kinase, BT, and higher values of triglycerides, also working at the same speed and distance with a lower HRmax and HRmed. Pull and helper horses had shown modifications of biomarkers. Furthermore, 15-min rest interval between races improved performance of helper horses as they used properly energy sources and cardiovascular function, respecting precepts of welfare.

Transition period produces changes in blood and body composition in mares / Período de transição produz alterações na composição do sangue e do corpo em éguas

Clinical and metabolic evaluation is necessary for the monitoring of pregnant and lactating mares, as they reflect the health of the animal. The body condition of the mare is an indicator of reproductive efficiency. The study aimed to determine the possible variations in body and metabolic condition in Mangalarga Marchador mares during the transition period. Forty-eight mares distributed in two groups were used: Maintenance Group (MG), composed of non-pregnant and non-lactating mares, and Transition Group (TG), formed by pregnant mares and who after delivery became lactating. Analyzes were performed in the times T-60, T-30 and T-15 before delivery, first six hours (T0) after delivery and T15, T30 and T60 days after delivery. MG was evaluated only at one time (T-60). Body weight and fat-free mass differed (P<0.05) between the groups. The percentage of fat was lower in MG. Mares had a higher fat percentage in TG at T-60 and T-30 times. There was a difference (P<0.05) in the amount of cholesterol between MG and TG (T0, T15 and T30). Triglycerides were different between the groups. TG showed higher concentrations of non-esterified fatty acids (P<0.05). There was a higher amount of glucose in TG (delivery and lactation) when compared to mares in MG. Changes in body condition and metabolic constituents occurred in the animals resulting from physiological adaptations of the transition period. The energetic components are the most affected from the transition period, with intense fat mobilization to supply the body demands.(AU)

Avaliações clínicas e metabólicas são necessárias para o monitoramento de éguas gestantes e lactantes, pois refletem a saúde do animal. A condição corporal da égua pode ser um indicador da eficiência reprodutiva. O estudo objetivou determinar as possíveis variações na condição corporal e metabólica em éguas Mangalarga Marchador durante o período de transição. Foram utilizadas 48 éguas distribuídas em dois grupos: Grupo em Manutenção (GM) composto por éguas não gestantes e não lactantes; Grupo em Transição (GT) formado por éguas gestantes e que após o parto tornaram-se lactantes. As análises foram realizadas nos tempos T-60, T-30 e T-15 antes do parto, primeiras seis horas (T0) após o parto e T15, T30 e T60 dias após o parto. O GM foi avaliado apenas uma vez (T-60). O peso corporal e a massa livre de gordura diferiram (P<0,05) entre os grupos. O percentual de gordura foi menor em GM. As éguas apresentaram maior porcentagem de gordura no GT no T-60 e no T-30. Houve diferença (P<0,05) na quantidade de colesterol entre GM e GT (T0, T15 e T30). Triglicérides foram diferentes entre os grupos. GT apresentou maiores concentrações de ácidos graxos não esterificados (P<0,05). Houve maior quantidade de glicose no GT (parto e lactação) quando comparada às éguas no GM. Mudanças na condição corporal e nos constituintes metabólicos ocorreram nos animais resultantes de adaptações fisiológicas do período de transição. Os componentes energéticos são os mais afetados neste período, existindo intensa mobilização de gordura para suprir demandas corporais.(AU)

Equine placenta expresses glutamine synthetase.

In most mammalian species the developing fetus utilizes large amounts of glutamine derived both from the maternal circulation and synthesized de novo in the placenta. The present study was designed to determine the role of the placenta in glutamine synthesis in the horse. The placentae from eight Standardbred mares were sampled immediately after parturition together with additional tissues obtained at necropsy from three Standbred mares during diestrous. Glutamine synthetase protein was detectable in the non-pregnant horn of the placenta in amounts similar to those seen in gluteus muscle, but the amount in the pregnant horn was two times greater than in the non-pregnant horn. Glutamine was the second most abundant amino acid in amniotic fluid at a concentration of 310 +/- 26 micromole/L with that of glycine being 535 +/- 48 micromole/L. The most abundant amino acids in placental tissue were glycine (3,732 +/- 194 micromole/Kg), glutamate (3,500 +/- 343 micromole/Kg) and glutamine (2,836 +/- 208 micromole/Kg). The results illustrate the importance of glutamine to the equine fetus and establish that the placenta, particularly the pregnant horn, has considerable capacity for glutamine synthesis.

Distribution of glutamine synthetase and an inverse relationship between glutamine synthetase expression and intramuscular glutamine concentration in the horse.

Glutamine plays important roles in the interorgan transport of nitrogen, carbon and energy but little is known about glutamine metabolism in the horse. In this study we determined the tissue distribution of glutamine synthetase expression in three Standardbred mares. Expression of glutamine synthetase was highest in kidney and mammary gland, and relatively high in liver and adipose tissue. Expression was lower in gluteus muscle, thymus, colon and lung, and much lower in small intestine, pancreas and uterus. The pattern of glutamine synthetase expression in the horse is similar to that of other herbivores and it is likely that skeletal muscle, liver, adipose tissue and lungs are the major sites of net glutamine synthesis in this species. Expression did not differ between adipose tissue depots but did vary between different muscles. Expression was highest in gluteus and semimembranous muscles and much lower in diaphragm and heart muscles. The concentration of intramuscular free glutamine was inversely correlated with expression of glutamine synthetase (r=-0.81, p=0.0017). The concentration of free glutamine was much higher in heart muscle (21.6+/-0.9 micromol/g wet wt) than in gluteus muscle (4.19+0.33 micromol/g wet wt), which may indicate novel functions and/or regulatory mechanisms for glutamine in the equine heart.

Exercise-induced alterations in plasma concentrations of ghrelin, adiponectin, leptin, glucose, insulin, and cortisol in horses.

Six Standardbred (STB) mares (11+/-2 years, 521+/-77 kg; means+/-SD) performed an exercise trial (EX) where they underwent an incremental exercise test (GXT) as well as a parallel control trial (CON) to test the hypothesis that short-term, high intensity exercise would alter plasma concentrations of glucose, leptin, adiponectin, ghrelin, insulin and cortisol. Plasma samples were taken before (0 min), during (last 10s at 6, 8m/s, and the velocity eliciting VO(2max)), and after exercise (2, 10, 30, 60 min; 12 and 24h post-GXT). A second set of blood samples was collected before and after an afternoon meal given at 1515 h (at 1500, 1514, 1530, and 1545 h). Data were analyzed using ANOVA for repeated measures and Tukey's test. During the GXT, there were no changes (P>0.05) in the plasma concentrations of glucose, leptin, adiponectin or ghrelin. However, there was a 29% increase (P<0.05) in mean plasma cortisol concentration and a 35% decrease (P<0.05) in mean plasma insulin concentration. Substantial increases (P<0.05) in the mean plasma concentrations of glucose and cortisol of 36% and 102%, respectively, were seen in the EX trial during the first 60 min post-GXT. Plasma leptin concentration, measured at the 24h post-GXT time point, was 20% lower (P<0.05) during the EX trial compared with the parallel time point in the standing control (CON) trial. Plasma ghrelin concentration was 37% lower (P<0.05) in the EX trial compared with CON before and after the afternoon meal, but was 43% higher (P<0.05) 12h post-GXT. There were no differences between EX and CON for plasma concentrations of insulin or adiponectin during recovery. It was concluded that short-term high intensity exercise alters plasma leptin and ghrelin concentrations in STB mares post-exercise, which may signal the exercised animals to alter energy intake.

Plasma leptin, ghrelin and adiponectin concentrations in young fit racehorses versus mature unfit standardbreds.

Concentrations of hormones related to energy homeostasis may differ between populations with varied body compositions, acting as signals to increase or decrease energy intake and/or expenditure. How these parameters correlate with body composition in horses and how they vary in fit (F) versus unfit (UF) Standardbred racehorses is unclear. The purpose of this study was to test the hypothesis that plasma concentrations of glucose (GLU), insulin (INS), cortisol (CORT), ghrelin (GHRL), adiponectin (ADIP) and leptin (LEP) would be correlated with body composition and differ in fit (F) versus unfit (UF) horses. Fasting plasma samples were taken from 12 unfit (11 +/- 2 years, 521 +/- 77 kg; mean +/- SD) and 34 fit (4 +/- 2 years, 475 +/- 83 kg) Standardbred horses. GHRL, LEP, ADIP, INS and CORT concentrations were measured using radioimmunoassay. GLU concentration was measured using colorometric kits. Body composition data included body weight, body condition score (BCS), and percent fat (%fat) calculated using rump fat thickness measured ultrasonically and the Westervelt equation. Data were analyzed using Pearson Product moment and Student's t tests. There were no differences (P>0.05) between F and UF horses for the plasma concentrations of CORT (69 +/- 14 versus 76 +/- 23 microg/dL), INS (7.2 +/- 3.5 versus 7.1 +/- 1.8 microIU/mL) or GLU (90 +/- 6 versus 86 +/- 7 mg/dL). Plasma GHRL and ADIP concentrations were greater (P<0.05) in F versus UF horses (54 +/- 27 versus 33 +/- 17 pg/mL and 1820 +/- 276 versus 1333 +/- 249 ng/mL, respectively), while plasma LEP was lower in F versus UF (1.0 +/- 0.6 versus 4.4 +/- 2.4 ng/mL, P<0.001). BCS and %fat were lower in F versus UF horses (4.8 +/- 0.3 versus 6.7 +/- 0.5 and 11.9 +/- 1.6 versus 15.4 +/- 2.5%, respectively), with no correlation between %fat and GHRL (-0.12, P>0.05), although there was a positive correlation between %fat and LEP (+0.72, P<0.05), and a negative correlation between %fat and ADIP (-0.40, P<0.05). The data show that in comparing fit and unfit horses, there are variations in body composition as well as concurrent and substantial differences in the concentrations of hormones, cytokines, and other parameters related to the control of appetite and feed intake.

Novel findings regarding Glut-4 expression in adipose tissue and muscle in horses--a preliminary report.

One of the hallmarks of insulin resistance is a reduction in glucose transporter-4 (Glut-4) expression in adipose tissue but not in skeletal muscle. However, while Glut-4 has been demonstrated in skeletal and cardiac muscles in horses it has not been demonstrated in adipose tissue. The initial objectives of the present study were: (1) to test the hypothesis that Glut-4 expression would vary between selected key skeletal muscles; (2) to test the hypothesis that it would also vary between representative adipose tissue depots, and (3) to see whether expression would be greater in adipose tissue compared to muscle. Glut-4 expression was determined by Western blot using samples obtained from post mortem biopsies obtained from four muscles (gluteus medius, semitendinosus, heart, and diaphragm), and four adipose tissues (subcutaneous, retroperitoneal, mesenteric, and omental) in three horses. There were no differences (P>0.05) in Glut-4 protein expression between the muscles sampled. Likewise there were no differences (P>0.05) in Glut-4 protein expression between fat depots. There was a significant difference (P=0.03) when pooled means for Glut-4 expression in muscle (58.8+/-2.5 densitometry units) were compared with adipose tissue (115.8+/-15.7). This difference in Glut-4 expression in these two tissues with distinctly different metabolic reasons for taking up glucose may warrant further investigation to see if there are more pronounced differences in Glut-4 expression in muscle and adipose tissue in various populations of horses.