Candidate genes for physical performance in the horse

Intense selection for speed, endurance or pulling power in the domestic horse (Equus caballus) has resulted in a number of adaptive changes in the phenotype required for elite athletic performance. To date, studies in humans have revealed a large number of genes involved in elite athletic performance, but studies in horses are rare. The horse genome assembly and bioinformation tools for genome analyses have been used to compare human performance genes with their equine orthologues, both to retrieve pathways for these genes and to investigate their chromosomal distribution. In this review, 28 candidate genes for equine performance are presented that have polymorphisms associated with human elite athletic performance and may have impact on athletic performance in horses. A significant accumulation of candidate genes was found on horse chromosomes 4 and 12. Genes involved in pathways for focal adhesion, regulation of actin cytoskeleton, neuroactive ligand-receptor interaction, and calcium signalling were over-represented. Genome-wide association studies for athletic performance in horses may benefit from the strong conserved synteny of the chromosomal arrangement of genes in humans and horses.     

Molecular cloning of horse Hsp90 cDNA and its comparative analysis with other vertebrate Hsp90 sequences

Heat shock protein 90 (Hsp90), a molecular chaperone, is ubiquitous and involved in numerous cellular processes. To contribute to the relatively small collection of vertebrate Hsp90 sequences in the gene data bank, we cloned and sequenced horse (Equus caballus) Hsp90 alpha and beta cDNAs. This enabled identification of horse-specific primers for development of a convenient PCR-based method that could monitor horse stress tolerance. We analyzed the sequence data comparatively and phylogenetically with other Hsp90 cDNA sequences, and identified vertebrate-specific and isoform-specific conserved regions to facilitate future molecular investigations of Hsp90 functions. We found 4 highly conserved regions to vertebrate Hsp90 exclusively and 27 amino acids conserved among but differing between Hsp90 alpha and Hsp90 beta sequences. Protein-based phylogenetic trees revealed high conservation between mammal species within Hsp90 alpha and beta clusters. Comparison of nucleotide and amino acid substitution levels suggests that horse Hsp90 beta has undergone strong purifying selection, while rat Hsp90 beta and hamster Hsp90 alpha have been positively selected. Surprisingly, fish Hsp90 alpha genes clearly clustered with Hsp90 beta genes, and no distinct placement of fish Hsp90 alpha protein was found. The Hsp90 alpha isoform is apparently the result of beta gene duplication. Our results highlight the importance of organism- and isoform-specific Hsp90 functional analyses in describing the role of Hsp90 in cells.     

Endurance Training Has Little Impact on Mineral Content of the Third Metacarpus in Two-Year-Old Arabian Horses

Although previous research suggests that short bouts of high-speed exercise will increase bone mass in horses, little research has been conducted to determine the impact of endurance exercise on bone. Although many in the equine industry believe that months of slow training will increase bone strength, we hypothesized that endurance training would not alter bone mineral content as determined through optical density. Eleven 2-year-old Arabians were split into two groups, Exercised (E, 6 geldings) or Control (C, 5 fillies), with radiographs taken on day 0 for estimation of bone mineral content by radiographic bone aluminum equivalence (RBAE). The E horses were then trained on a high-speed treadmill for 90 days; training consisted of walking (1.6 m/second), trotting (4 m/second) and cantering (8 m/second) at increasing distances until the target of 60 km/day was met. On day 90, E horses were confined to 9 × 14 m dry lots and placed on a regular exercise schedule, including a 60-km endurance test once every 3 weeks. The C horses remained on pasture throughout the study. On day 162, radiographs were made again on all 11 horses. No differences between treatments, days, or interactions were noted in any cortex or the total RBAE, suggesting that endurance exercise does little to alter bone optical density compared with free-choice exercise on pasture.     

The Effect of Different Physical Exercise on Plasma Leptin,Cortisol, and Some Energetic Parameters Concentrations in Purebred Arabian Horses

Leptin is a hormone that coordinates food intake, energy expenditure, and metabolic rate; it is, however, unknown how exercise influences plasma leptin concentration in horses. The aim of the study was to evaluate the exercise-induced changes in plasma leptin concentration in purebred Arabian horses competing in races and endurance rides. A total number of 26 horses (12 purebred Arabian racehorses aged 3–5 years and 14 endurance horses aged 7–14 years) were studied during performing routine physical exercise. From each horse, blood samples were collected at rest and immediately after the exercise session. Concentrations of plasma leptin, cortisol, lactic acid, uric acid, triacylglycerols, glycerol and free fatty acids were determined. Exercise-induced increase in cortisol and free fatty acid values were higher in endurance horses than in racehorses. Neither endurance nor race exercises influenced the plasma leptin concentration. Further research is needed to elucidate the effects of different types of exercise on leptin synthesis and secretion in horses.     

蒙古马高负荷运动训练前后COX4I2、FABPpm和CAV-1基因表达量的研究

为研究蒙古马高负荷运动训练前后COX4I2、FABPpm和CAV-1基因表达量的影响,实验选择3匹5岁雌性蒙古马,采用qRT-PCR方法检测为期4个月的强度递增高负荷运动训练后蒙古马臀中肌3种基因在mRNA水平的表达变化。结果表明:经训练后蒙古马肌肉中COX4I2和FABPpm基因表达量均显著升高(P<0.05);CAV-1基因表达量也有升高(P>0.05)。可见,COX4I2、FABPpm和CAV-1基因与蒙古马的运动能力存在密切关系。     

Changes in blood constituents accompanying exercise in polo horses

There have been several studies of biochemical changes in horses doing intense exercise such as Thoroughbred and Standardbred racehorses and in horses performing exercise over a long period of time such as endurance horses and three-day eventing horses, but we are not aware of studies with polo horses. Blood samples were taken from 18 polo horses at rest, immediately after playing 2 chukkers of indoor polo, and after a 15 minute rest period. Each horse was studied at 2 different games. The blood samples were analyzed for lactic acid, protein, sodium, potassium, chloride, calcium, phosphorus, HCO-3, PCO2, hemoglobin, packed cell volume, and pH. Samples taken immediately after playing polo had significant increases in lactic acid, protein, sodium, hemoglobin, packed cell volume, and pH, and significant decreases in chloride, calcium, PCO2, and HCO-3. Pulse and respiration were significantly increased. After a 15 minute rest period, there was a significant decrease in potassium. The HCO-3 was lower immediately after playing, but was above the resting value after 15 minutes. It was concluded that the changes after exercise are similar in some aspects to those reported for horses performing intense exercise such as racehorses, and in some aspects to those reported for horses performing prolonged exercise such as three-day event horses and endurance horses. Horses playing indoor polo develop a high plasma lactic acid, but with alkalemia, and could be used as a model to study this condition.     

Clinical Applications of the Renin-Angiotensin-Aldosterone-Vasopressin Axis in the Horse and Future Directions for Research

The renin–angiotensin–aldosterone–vasopressin axis (RAAV) is known to play significant roles in preserving hemodynamic stability in response to changes in blood volume, blood pressure, electrolytes, and water. In the previous years, some studies focused on RAAV in the horse, showing substantial future clinical and research applications. Early studies assessed the response of this axis to different types of exercise (increasing intensity exercise vs. endurance). Aldosterone and vasopressin concentrations were measured in horses with mild dehydration induced by endurance exercise in comparison with horses suffering from exhaustion and metabolic problems after prolonged exercise. More recently, the neurohumoral response to heart failure, mainly in congestive states, has received special attention. The activation of the RAAV in congestive heart failure has important prognosis applications and inhibitors of this axis have been clinically studied, as the angiotensin-converting enzyme inhibitors. However, inhibitors of renin, aldosterone, and vasopressin have not been studied in horses. Additionally, the investigation of the changes in blood pressure during dehydration, sepsis, and endotoxemia in foals and adult horses might show important applications in the treatment of states that induce modifications in blood pressure. Inappropriately low RAAV activity has been detected in human patients with prolonged vasodilatory shock, and exogenously administered vasopressin could be used as a potent vasopressor agent to stabilize cardio-circulatory function. Similarly, it might be administered to septic foals for similar purposes. Finally, the RAAV axis also has important implications in the development as well as in the treatment of the chronic renal failure, although it has not been investigated in horses.     

Heart rate variability after horse trekking in leading and following horses

Horse trekking (HT) is having a stroll on a horse along a walking trail in a forest, field, and/or sandy beach. Generally in HT, horses exercise in tandem line outside the riding facilities. Because the leading horse will be confronted with stressors in the forefront, we hypothesized that the leading horse shows higher stress responses than the following one. In order to verify the hypothesis, we compared short‐term stress responses between each position in six horses. Exercise consisted of 15 min of ground riding and 45 min of HT with walking and trotting. Heart rate variability was analyzed for 5 min at 30, 60, and 90 min after the exercising period. There was no significant difference in heart rate during exercise between leading and following positions. The high frequency / low frequency power band of heart rate variability, an index of sympathetic nervous activity, after exercise, tended to be higher in the leading position than following one (P < 0.1). The result in this study can suggest that the leading horse was in a higher stressed state than the following horse after HT.     

蒙古家马和蒙古野马线粒体细胞色素b基因遗传多样性及系统进化分析

通过对马的线粒体细胞色素b（Cytb）基因序列的遗传变异分析,来探讨蒙古家马、蒙古野马和驴之间的亲缘关系和遗传多样性。对各类马和驴样本 mtDNA Cytb 基因进行PCR扩增和测序,运用现代分子生物学软件进行数据处理。结果共检测到15个单倍型,90个突变位点,单倍型多样性（Hd）为0.9356,核苷酸多样性（Pi）为0.01022,平均核苷酸差异数（K）为8.966,各群体间的遗传多样性相差不大。系统发育树和NETwork图中每一分支都至少包含2个群体的信息。4个类型蒙古家马总体上具有较高的遗传多态性,蒙古家马与蒙古野马之间的亲缘关系较近, 且出现数个分支,蒙古家马分布在各独立的进化支中,说明蒙古家马母系来源非常广泛或经过多次驯化。     

Contribution to our knowledge of the physiology and metabolism of endurance horses

The functional and metabolic processes of the endurance horse are discussed, based on personal experience and the scientific literature. Research on the energetic and physiological aspects of prolonged effort is reviewed, as regards the performance structure, described by Neumann, for human physiology.Experimental data from our previous surveys on endurance horses, competing at different levels, are also presented and discussed.The results on amino acid levels in blood, and related metabolic pathways, during endurance events, lead to speculation on the effects of the race distance on metabolic processes during long-lasting low-intensity exercise. In fact, the event distance has a significant influence on amino acid mobilization and their use as energy sources. In human athletes, the disappearance of some amino acids from blood circulation is linked to the onset of central fatigue. The effect could be similar in endurance horses; therefore, the correct intake of amino acids, and a proper feeding strategy, could improve performance.Data are also provided on dehydration and oxidative stress associated with endurance work.     

Genetic parameters of performance traits in horse endurance races

The objective of the study was to find a heritable trait measuring ability in endurance races from competition results in order to select endurance horses from them. The results of 90–160 km endurance races in France from 2002 to 2004 were available including 2795 horses, 3.2 races per horse, from which 2264 had a speed measured, i.e. 5252 measurements of speed and 9059 of placing (0 or 1 when the horse completed the race). The model included fixed effects of sex, age, month of birth (not significant), region of birth, breed, and random effects of permanent environmental effects and animal effect (20,854 horses). Heritabilities were 0.28 and 0.06 (0.10 on underlying scale) respectively for speed and placing. Repeatabilities were respectively 0.44 and 0.07 (0.12). The genetic correlation was 0.87. No genetic differences were found between the results in small distance races (90 km) and long distance races (120–160 km). Additional data on young horses in specific races (20–60 km) with controlled speed did not give a precocious criterion (genetic correlation between speed in adult races and maximum level of distance and difficulty in the young horse equal to 0.07) for selection. In conclusion, breeding values should be calculated from speed and placing in races ≥ 90 km. A higher genetic progress is expected than for other equestrian disciplines since heritability is considerably higher for a trait measured in a single event.     

Nucleotide structure of equine platelet-derived growth factor-A and -B and expression in horses with induced acute tendinitis

OBJECTIVE: To characterize the nucleotide sequence of equine platelet-derived growth factor (PDGF)-A and -B and analyze temporal expression of these genes in equine tendon after induced tendinitis injury. Animals-18 mature horses. PROCEDURES: Genes for equine PDGF-A and -B were reverse transcribed and sequenced from synovial tissue mRNA obtained from a 3-year-old horse. Collagenase-induced lesions were created in the tensile region of the superficial digital flexor tendon in 14 horses; 3 horses served as uninjured control animals. Tendons were harvested and total RNA was isolated from experimental horses 1, 2, 4, 8, and 24 weeks after collagenase injection. Temporal gene expression for PDGF-A and -B was determined by use of quantitative PCR analysis. RESULTS: Equine PDGF-A shared 83.8% sequence and 87.5% peptide homology with human PDGF-A, with a discrepancy of 70 bp from the human sequence. Equine PDGF-B was similar in length to the human gene, sharing 90.3% and 91.7% nucleotide and peptide identity, respectively. Expression of PDGF-A mRNA in collagenase-induced tendinitis lesions was unchanged, compared with expression for normal control tendon, and remained steady throughout the 24-week study. Expression of PDGF-B mRNA decreased over time, and the expression at 24 weeks was significantly reduced, compared with expression in normal and acutely injured tendon. CONCLUSIONS AND CLINICAL RELEVANCE: Injured tendon mounts a minimal constitutive PDGF-A or -B mRNA response. Serial exogenous treatment with either PDGF isoform within the first 2 to 4 weeks after tendon injury may bolster the meager PDGF paracrine-autocrine intrinsic response to injury.     

Long term intensive exercise training leads to a higher plasma malate/lactate dehydrogenase (M/L) ratio and increased level of lipid mobilization in horses

Continuous high intensity training may induce alterations to enzyme activities related to glucose and lipid metabolism in horses. In our study, five Thoroughbred race horses (3 male and 2 female, avg age=5 yrs old) were compared against five riding horses (1 male, 1 female, 3 gelding, avg age=13 yrs old) in terms of energy metabolism, by examining plasma malate (MDH) and lactate (LDH) dehydrogenase activities and M/L ratio. MDH is involved in NADH and ATP generation, whereas LDH can convert NADH back into NAD(+) for ATP generation. An increase in plasma M/L ratio can reflect heightened energy metabolism in the liver and skeletal muscle of horses adapted to continuous intensive exercise. Moreover, plasma lipid metabolism analytes (adiponectin, NEFA, total cholesterol (T-Cho), and triglycerides (TG)) can reflect changes to lipolysis rate, which can also indicate a change in energy metabolism. Overall, race horses demonstrated increased MDH and LDH activity in plasma (4x and 2x greater, respectively), in addition to a plasma M/L ratio twice as high as that of riding horses (2.0 vs 1.0). In addition, race horses also demonstrated significantly higher levels of plasma NEFA (50% greater), TG (2x greater), and T-Cho (20% greater) as compared to riding horses. Therefore, race horse muscles may have adapted to prolonged high intensity endurance exercise by gaining a higher oxidative capacity and an increased capacity for fat utilization as an energy source, resulting in heightened energy metabolism and increased rate of lipid mobilization.     

An acute outbreak of equine dysautonomia (equine grass sickness) in a group of eight Przewalski's horses (Equus ferus [caballus] przewalskii)

Equine grass sickness (EGS) is a neurodegenerative disease affecting grazing equids of which a single case of the chronic clinical presentation has previously been reported in a Przewalski's horse (Equus ferus [caballus] przewalskii). A group of 8 Przewalski's horses were moved to a new enclosure, recently vacated by a group of 4 Eastern kiang (Equus kiang holdereri) that showed no evidence of disease. After 23 days the first Przewalski's horse showed clinical signs of acute EGS including flank sweating, belly kicking, rapid loss of body condition, cessation of faecal passage, nasogastric reflux and mouthing water. It was subjected to euthanasia within 48 h due to lack of therapeutic response. Within 24 h of this first case developing clinical signs, a further 5 Przewalski's horses showed similar clinical signs of acute EGS and were subjected to euthanasia. Post mortem examinations confirmed acute EGS, with all animals demonstrating typical chromatolysis, cytoplasmic hypereosinophilia, cellular swelling, vacuolation, pyknosis and loss of nuclei in approximately 90% of neurones in the cranial cervical and cranial mesenteric ganglia and myenteric and submucosal plexi of the ileum. Two Przewalski's horses within the group showed no clinical signs of disease. No single pathogen was identified as the causal agent, but the epidemiological pattern of the outbreak was typical for that previously reported for acute EGS in domestic equids. All affected animals and the 2 surviving Przewalski's horses had low antibody titres to Clostridium botulinum type C. This is the first report of acute EGS in a herd of Przewalski's horses.     

The opioid haemorphin-7 in horses during low-speed and high-speed treadmill exercise to fatigue

The opioid neuropeptide haemorphin-7 was measured, by immunoreactivity, in Standardbred horses during low-speed (7 m/s) and high-speed (10 m/s) endurance exercises, lasting 49-58 and 12-16 min respectively. In parallel, heart rate, muscle temperature and plasma lactate concentrations were measured. The profile of the low-speed exercise showed significantly increased heart rate after 10 min [154 beats per minute (bpm)]. After the exercise, muscle temperature (42.1 degrees C) and plasma lactate (4.8 mmol/l) were significantly increased. The profile of the high-speed exercise was comparatively characterized by a higher increase of heart rate after 5 min (194 bpm) and higher increases of muscle temperature (43.2 degrees C) and lactate levels (15.8 mmol/l) after the exercise. The horses were probably exhausted by glycogen depletion in the low-speed exercise and by muscle pH decrease in the high-speed exercise. Haemorphin-7 increased significantly during the high-speed exercise (274.8 fmol/ml) but not during low speed (108.3 fmol/ml), coincident with the results of lactate. These results suggest that plasma haemorphin-7 is measurable in the horse by immunoreactivity, and that intense exercise stimulates release of this opioid. Such endogenous opioids are most likely involved in regulatory functions associated with pain, physical effort, inflammation, and blood pressure variation in horses, as have been established in other species.     

Differential responses of Equus caballus and Equus asinus to infection with two pathogenic strains of equine infectious anemia virus

Most in vivo studies with equine infectious anemia virus (EIAV) have been performed in horses and ponies (Equus caballus) with little published information available detailing the clinical responses of donkeys (Equus asinus) to infection with this virus. Consequently, donkeys were inoculated with two strains of EIAV (EIAV(PV) and EIAV(WY)) which have been documented to produce disease in E. caballus. Four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with EIAV(PV) and one horse (94-10) and one donkey (4) were infected with EIAV(WY). Although the horse and ponies all experienced clinical signs of disease, which in some cases were severe, the donkeys remained asymptomatic throughout a 365-day observation period, except for mild transient reductions in platelet counts. The results from serological assays, virus isolation from plasma and detection of plasma-associated viral RNA by RT-PCR, indicated that initial replication of EIAV(PV) and EIAV(WY) was lower in donkeys than in horses and ponies. This conclusion was confirmed using competitive RT-PCR, in which viral RNA levels in the plasma of EIAV(PV)-infected ponies was up to 100,000-fold higher than in infected donkeys during the first 20 days post-infection (dpi). Similar results were obtained in the EIAV(WY)-infected animals, in which viral RNA burdens in the donkey at 20 dpi were 1000-fold less than in the horse. However, infection of donkey and horse monocyte-derived macrophage cultures with EIAV(PV) demonstrated that these cells in vitro were equally susceptible to virus-induced cytopathic effects and yielded similar levels of progeny virus. This result suggests that factors other than host cell permissiveness mediate the clinical differences observed between horses and donkeys infected with EIAV(PV) or EIAV(WY).     

Duodenal Glands of the Pony (Equus caballus)

The ultrastructure and histochemistry of the duodenal glands of the pony (Equus caballus) was examined in four horses. Unlike that of most species except for the rabbit, the submucous glands of the horse contain two distinct cell types, serous and mucous. These cells are described.