Variation in salivary cortisol responses in yearling Thoroughbred racehorses during their first year of training.

Thoroughbred horses are bred for competitive racing and undergo intense training regimes. The maintenance of physical soundness and desirable behavioural characteristics are critical to the longevity of a racing career. Horses intended for Flat racing generally enter training as yearlings and undergo introductory training prior to exercise conditioning for racing. This period requires rapid adjustment to a novel environment. As a prey animal, a horse's 'fight-or-flight' response is highly adapted, in which a well-understood component of this response, the hypothalamic-pituitary-axis, is activated in response to a stress stimulus, releasing cortisol. In the Thoroughbred, a significant difference in salivary cortisol concentrations between pre- and post-first time ridden (i.e., first backing) by a jockey have previously been identified. Here, to test the hypothesis that salivary cortisol concentrations may be used to objectively detect individual variations in the acute physiological stress response we investigate individual variation in cortisol response to training milestones. Saliva samples were collected from a cohort of n = 96 yearling Flat racehorses, at the same training yard, across three timepoints at rest: before entering the training yard (n = 66), within three days of entry to the training yard (n = 67) and following 2-3 weeks in the training yard (n = 50). Salivary cortisol concentration was measured using an ELISA. There was no significant difference in cortisol concentration (ANOVA, P > 0.05) across the samples collected at timepoints at rest. Samples were also collected before and 30 minutes after exposure to three novel training events: first time long-reined (n = 6), first time backed by a jockey (n = 34), and first time ridden on the gallops (n = 10). Mean salivary cortisol concentration after all three novel training events was significantly higher than prior to the training event (Paired t-test, P <0.005). The ranges of post-event salivary cortisol concentration across all timepoints suggest individual variation in the measured stress response, reflecting individual differences in stress response to the early training environment. This measure may be used as an objective assessment of the stress response of Thoroughbred racehorses during training.

Integrative genomics analysis highlights functionally relevant genes for equine behaviour.

Behavioural plasticity enables horses entering an exercise training programme to adapt with reduced stress. We characterised SNPs associated with behaviour in yearling Thoroughbred horses using genomics analyses for two phenotypes: (1) handler-assessed coping with early training events [coping] (n = 96); and (2) variation in salivary cortisol concentration at the first backing event [cortisol] (n = 34). Using RNA-seq derived gene expression data for amygdala and hippocampus tissues from n = 2 Thoroughbred stallions, we refined the SNPs to those with functional relevance to behaviour by cross-referencing to the 500 most highly expressed genes in each tissue. The SNPs of high significance (q < 0.01) were in proximity to genes (coping - GABARAP, NDM, OAZ1, RPS15A, SPARCL1, VAMP2; cortisol - CEBPA, COA3, DUSP1, HNRNPH1, RACK1) with biological functions in social behaviour, autism spectrum disorder, suicide, stress-induced anxiety and depression, Alzheimer's disease, neurodevelopmental disorders, neuroinflammatory disease, fear-induced behaviours and alcohol and cocaine addiction. The strongest association (q = 0.0002) was with NDN, a gene previously associated with temperament in cattle. This approach highlights functionally relevant genes in the behavioural adaptation of Thoroughbred horses that will contribute to the development of genetic markers to improve racehorse welfare.

Selection signatures for local and regional adaptation in Chinese Mongolian horse breeds reveal candidate genes for hoof health.

BACKGROUND: Thousands of years of natural and artificial selection since the domestication of the horse has shaped the distinctive genomes of Chinese Mongolian horse populations. Consequently, genomic signatures of selection can provide insights into the human-mediated selection history of specific traits and evolutionary adaptation to diverse environments. Here, we used genome-wide SNPs from five distinct Chinese Mongolian horse populations to identify genomic regions under selection for the population-specific traits, gait, black coat colour, and hoof quality. Other global breeds were used to identify regional-specific signatures of selection. RESULTS: We first identified the most significant selection peak for the Wushen horse in the region on ECA23 harbouring DMRT3, the major gene for gait. We detected selection signatures encompassing several genes in the Baicha Iron Hoof horse that represent good biological candidates for hoof health, including the CSPG4, PEAK1, EXPH5, WWP2 and HAS3 genes. In addition, an analysis of regional subgroups (Asian compared to European) identified a single locus on ECA3 containing the ZFPM1 gene that is a marker of selection for the major domestication event leading to the DOM2 horse clade. CONCLUSIONS: Genomic variation at these loci in the Baicha Iron Hoof may be leveraged in other horse populations to identify animals with superior hoof health or those at risk of hoof-related pathologies. The overlap between the selection signature in Asian horses with the DOM2 selection peak raises questions about the nature of horse domestication events, which may have involved a prehistoric clade other than DOM2 that has not yet been identified.

Warmblood fragile foal syndrome causative single nucleotide polymorphism frequency in horses in Ireland.

BACKGROUND: Warmblood Fragile Foal Syndrome (WFFS) is an autosomal recessive disorder caused by a mutation in the procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) gene. Homozygosity for the mutation results in defective collagen synthesis which clinically manifests as the birth of non viable or still born foals with abnormally fragile skin. While the mutation has been identified in non Warmblood breeds including the Thoroughbred, to date all homozygous clinically affected cases reported in the scientific literature are Warmblood foals. The objective of this study was to investigate the carrier frequency of the mutation in the Thoroughbred and sport horse populations in Ireland. METHODS: A test was developed at the UCD School of Veterinary Medicine using real-time PCR to amplify the PLOD1 gene c.2032G > A variant. A subset of the samples was also submitted to an external laboratory with a licensed commercial WFFS genetic test. RESULTS: Warmblood Fragile Foal Syndrome genotyping was performed on hair samples from 469 horses representing 6 different breeds. Six of 303 (1.98%) sport horses tested and three of 109 (2.75%) Thoroughbreds tested were heterozygous for the WFFS polymorphism (N/WFFS). The WFFS polymorphism was not identified in the Standardbred, Cob, Connemara, or other pony breeds. CONCLUSIONS: The study identified a low frequency of the WFFS causative mutation in sport horses and Thoroughbreds in Ireland, highlighting the importance of WFFS genetic testing in order to identify phenotypically normal heterozygous carriers and to prevent the birth of nonviable foals.

Inspiratory muscle training in young, race-fit Thoroughbred racehorses during a period of detraining.

Although inspiratory muscle training (IMT) is reported to improve inspiratory muscle strength in humans little has been reported for horses. We tested the hypothesis that IMT would maintain and/or improve inspiratory muscle strength variables measured in Thoroughbreds during detraining. Thoroughbreds from one training yard were placed into a control (Con, n = 3 males n = 7 females; median age 2.2±0.4 years) or treatment group (Tr, n = 5 males, n = 5 females; median age 2.1±0.3 years) as they entered a detraining period at the end of the racing/training season. The Tr group underwent eight weeks of IMT twice a day, five days per week using custom-made training masks with resistance valves and an incremental threshold of breath-loading protocol. An inspiratory muscle strength test to fatigue using an incremental threshold of breath-loading was performed in duplicate before (T0) and after four (T1) and eight weeks (T2) of IMT/no IMT using a custom-made testing mask and a commercial testing device. Inspiratory measurements included the total number of breaths achieved during the test, average load, peak power, peak volume, peak flow, energy and the mean peak inspiratory muscle strength index (IMSi). Data were analysed using a linear mixed effects model, P≤0.05 significant. There were no differences for inspiratory measurements between groups at T0. Compared to T0, the total number of breaths achieved (P = 0.02), load (P = 0.003) and IMSi (P = 0.01) at T2 had decreased for the Con group while the total number of breaths achieved (P<0.001), load (P = 0.03), volume (P = 0.004), flow (P = 0.006), energy (P = 0.01) and IMSi (P = 0.002) had increased for the Tr group. At T2 the total number of breaths achieved (P<0.0001), load (P<0.0001), volume (P = 0.02), energy (P = 0.03) and IMSi (P<0.0001) were greater for the Tr than Con group. In conclusion, our results support that IMT can maintain and/or increase aspects of inspiratory muscle strength for horses in a detraining programme.

Selection in Australian Thoroughbred horses acts on a locus associated with early two-year old speed.

Thoroughbred horse racing is a global sport with major hubs in Europe, North America, Australasia and Japan. Regional preferences for certain traits have resulted in phenotypic variation that may result from adaptation to the local racing ecosystem. Here, we test the hypothesis that genes selected for regional phenotypic variation may be identified by analysis of selection signatures in pan-genomic SNP genotype data. Comparing Australian to non-Australian Thoroughbred horses (n = 99), the most highly differentiated loci in a composite selection signals (CSS) analysis were on ECA6 (34.75-34.85 Mb), ECA14 (33.2-33.52 Mb and 35.52-36.94 Mb) and ECA16 (24.28-26.52 Mb) in regions containing candidate genes for exercise adaptations including cardiac function (ARHGAP26, HBEGF, SRA1), synapse development and locomotion (APBB3, ATXN7, CLSTN3), stress response (NR3C1) and the skeletal muscle response to exercise (ARHGAP26, NDUFA2). In a genome-wide association study for field-measured speed in two-year-olds (n = 179) SNPs contained within the single association peak (33.2-35.6 Mb) overlapped with the ECA14 CSS signals and spanned a protocadherin gene cluster. Association tests using higher density SNP genotypes across the ECA14 locus identified a SNP within the PCDHGC5 gene associated with elite racing performance (n = 922). These results indicate that there may be differential selection for racing performance under racing and management conditions that are specific to certain geographic racing regions. In Australia breeders have principally selected horses for favourable genetic variants at loci containing genes that modulate behaviour, locomotion and skeletal muscle physiology that together appear to be contributing to early two-year-old speed.

Genomic inbreeding trends, influential sire lines and selection in the global Thoroughbred horse population.

The Thoroughbred horse is a highly valued domestic animal population under strong selection for athletic phenotypes. Here we present a high resolution genomics-based analysis of inbreeding in the population that may form the basis for evidence-based discussion amid concerns in the breeding industry over the increasing use of small numbers of popular sire lines, which may accelerate a loss of genetic diversity. In the most comprehensive globally representative sample of Thoroughbreds to-date (n = 10,118), including prominent stallions (n = 305) from the major bloodstock regions of the world, we show using pan-genomic SNP genotypes that there has been a highly significant decline in global genetic diversity during the last five decades (FIS R2 = 0.942, P = 2.19 × 10-13; FROH R2 = 0.88, P = 1.81 × 10-10) that has likely been influenced by the use of popular sire lines. Estimates of effective population size in the global and regional populations indicate that there is some level of regional variation that may be exploited to improve global genetic diversity. Inbreeding is often a consequence of selection, which in managed animal populations tends to be driven by preferences for cultural, aesthetic or economically advantageous phenotypes. Using a composite selection signals approach, we show that centuries of selection for favourable athletic traits among Thoroughbreds acts on genes with functions in behaviour, musculoskeletal conformation and metabolism. As well as classical selective sweeps at core loci, polygenic adaptation for functional modalities in cardiovascular signalling, organismal growth and development, cellular stress and injury, metabolic pathways and neurotransmitters and other nervous system signalling has shaped the Thoroughbred athletic phenotype. Our results demonstrate that genomics-based approaches to identify genetic outcrosses will add valuable objectivity to augment traditional methods of stallion selection and that genomics-based methods will be beneficial to actively monitor the population to address the marked inbreeding trend.

Determination of reference intervals for umbilical cord arterial and venous blood gas analysis of healthy Thoroughbred foals.

Although umbilical cord blood gas analysis is considered the best way to assess in utero oxygenation in human neonates, there is limited evaluation of this method in equine neonatology. Our objectives were to assess the practicality of obtaining umbilical cord blood gas samples in the field and to determine umbilical cord arterial and venous blood gas reference intervals (RI) for healthy, newborn foals. Thoroughbred foals >320 days gestation from healthy mares with uneventful pregnancies at one stud farm were evaluated. All parturitions were observed, with paired umbilical arterial and venous whole-blood samples obtained immediately following parturition for blood gas and lactate concentrations measured in duplicate. Apgar scores were assigned immediately and 10 min after birth, with all foals subsequently examined on days 1-28 to monitor for development of perinatal asphyxia syndrome. Foals were excluded from analysis based on abnormalities of stage 2 labour, Apgar scores and gross and histological placental assessment. Data was analysed using a Student's t-test, Pearson's correlation and the Robust method with P ≤ 0.05 significant. Umbilical cord samples were simple to obtain with minimal disruption to the foaling environment. Of the n = 34 foals assessed, n = 7 were excluded based on premature placental separation deliveries. The mean time for stage 2 labour and blood gas analysis after parturition was 17.3 ±â€¯5.1 min and 5.0 ±â€¯2.3 min, respectively. RI were identified for umbilical arterial and venous pH (7.19-7.42 vs. 7.34-7.44), PO2 (15.5-48.39 mmHg vs. 16.6-52.7 mmHg), PCO2 (49.5-82.29 mmHg vs. 45.4-63.1 mmHg), SO2 (9.19-76.89% vs. 39.9-84.88%), bicarbonate (27.3-38.7 mmol/l vs. 27.7-37.8 mmol/l), base excess (0.36-12.9 mmol/l vs. 1.97-13.1 mmol/l), TCO2 (28.99-40.3 mmHg vs. 29.0-39.5 mmHg) and lactate (1.4-7.3 mmol/l vs. 1.3-4.9 mmol/l). Umbilical arterial samples had lower pH (P < 0.0001), PO2 (P = 0.002) and SO2 (P < 0.0001) and higher PCO2 (P < 0.0001) and lactate (P < 0.0001) than venous samples. The initial Apgar score was positively correlated to umbilical arterial SO2 (r = 0.4, P = 0.05) and negatively with umbilical arterial TCO2 (r = -0.6, P = 0.004). Overall, umbilical cord sampling was simple and minimally disruptive, with RI obtained for blood gas measurements. RI for umbilical blood gas measurements from a larger population of healthy and unhealthy foals is required to evaluate the accuracy of this method for assessing in utero oxygenation.

Skeletal muscle mitochondrial bioenergetics and associations with myostatin genotypes in the Thoroughbred horse.

Variation in the myostatin (MSTN) gene has been reported to be associated with race distance, body composition and skeletal muscle fibre composition in the horse. The aim of the present study was to test the hypothesis that MSTN variation influences mitochondrial phenotypes in equine skeletal muscle. Mitochondrial abundance and skeletal muscle fibre types were measured in whole muscle biopsies from the gluteus medius of n = 82 untrained (21 ± 3 months) Thoroughbred horses. Skeletal muscle fibre type proportions were significantly (p < 0.01) different among the three MSTN genotypes and mitochondrial content was significantly (p < 0.01) lower in the combined presence of the C-allele of SNP g.66493737C>T (C) and the SINE insertion 227 bp polymorphism (I). Evaluation of mitochondrial complex activities indicated higher combined mitochondrial complex I+III and II+III activities in the presence of the C-allele / I allele (p ≤ 0.05). The restoration of complex I+III and complex II+III activities following addition of exogenous coenzyme Q1 (ubiquinone1) (CoQ1) in vitro in the TT/NN (homozygous T allele/homozygous no insertion) cohort indicated decreased coenzyme Q in these animals. In addition, decreased gene expression in two coenzyme Q (CoQ) biosynthesis pathway genes (COQ4, p ≤ 0.05; ADCK3, p ≤ 0.01) in the TT/NN horses was observed. This study has identified several mitochondrial phenotypes associated with MSTN genotype in untrained Thoroughbred horses and in addition, our findings suggest that nutritional supplementation with CoQ may aid to restore coenzyme Q activity in TT/NN horses.

Equine skeletal muscle adaptations to exercise and training: evidence of differential regulation of autophagosomal and mitochondrial components.

BACKGROUND: A single bout of exercise induces changes in gene expression in skeletal muscle. Regular exercise results in an adaptive response involving changes in muscle architecture and biochemistry, and is an effective way to manage and prevent common human diseases such as obesity, cardiovascular disorders and type II diabetes. However, the biomolecular mechanisms underlying such responses still need to be fully elucidated. Here we performed a transcriptome-wide analysis of skeletal muscle tissue in a large cohort of untrained Thoroughbred horses (n = 51) before and after a bout of high-intensity exercise and again after an extended period of training. We hypothesized that regular high-intensity exercise training primes the transcriptome for the demands of high-intensity exercise. RESULTS: An extensive set of genes was observed to be significantly differentially regulated in response to a single bout of high-intensity exercise in the untrained cohort (3241 genes) and following multiple bouts of high-intensity exercise training over a six-month period (3405 genes). Approximately one-third of these genes (1025) and several biological processes related to energy metabolism were common to both the exercise and training responses. We then developed a novel network-based computational analysis pipeline to test the hypothesis that these transcriptional changes also influence the contextual molecular interactome and its dynamics in response to exercise and training. The contextual network analysis identified several important hub genes, including the autophagosomal-related gene GABARAPL1, and dynamic functional modules, including those enriched for mitochondrial respiratory chain complexes I and V, that were differentially regulated and had their putative interactions 're-wired' in the exercise and/or training responses. CONCLUSION: Here we have generated for the first time, a comprehensive set of genes that are differentially expressed in Thoroughbred skeletal muscle in response to both exercise and training. These data indicate that consecutive bouts of high-intensity exercise result in a priming of the skeletal muscle transcriptome for the demands of the next exercise bout. Furthermore, this may also lead to an extensive 're-wiring' of the molecular interactome in both exercise and training and include key genes and functional modules related to autophagy and the mitochondrion.

MSTN genotypes in Thoroughbred horses influence skeletal muscle gene expression and racetrack performance.

Myostatin, encoded by the MSTN gene, is a member of the TGF-ß superfamily that regulates skeletal muscle development. A MSTN SNP significantly associated with Thoroughbred horse racing phenotypes has recently been identified as well as significant reductions in Thoroughbred skeletal muscle gene expression for three transcripts 400-1500 base pairs downstream of the MSTN gene following a period of training. Together, these findings indicate that MSTN genotypes may influence MSTN gene expression. To investigate this, MSTN mRNA expression was measured in biopsies from the middle gluteal muscle from 60 untrained yearling Thoroughbreds (C/C, n = 15; C/T, n = 28; T/T, n = 17) using two independent real-time qRT-PCR assays. MSTN gene expression was also evaluated in a subset (N = 33) of these animals using samples collected after a ten-month period of training. A significant association was observed between genotype and mRNA abundance for the untrained horses (assay I, P = 0.0237; assay II, P = 0.003559), with the C/C cohort having the highest MSTN mRNA levels, the T/T group the lowest levels and the C/T group intermediate levels. Following training, there was a significant decrease in MSTN mRNA (-3.35-fold; P = 6.9 × 10(-7) ), which was most apparent for the C/C cohort (-5.88-fold, P = 0.001). These data demonstrate the tight relationship between phenotype, genotype and gene expression at the MSTN gene in Thoroughbred racehorses.

MSTN genotype (g.66493737C/T) association with speed indices in Thoroughbred racehorses.

Sequence variation at the equine myostatin gene (MSTN) locus has previously been shown to have a singular genomic influence on optimum race distance in Thoroughbred racehorses. Myostatin, encoded by the MSTN gene, is a member of the TGF-ß superfamily that regulates skeletal muscle development in a range of mammalian species including the horse. In the Thoroughbred, the C-allele at the g.66493737C/T SNP has been found at significantly higher frequency in subgroups of the population that are suited to fast, short distance, sprint races and also influences body composition phenotypes. We investigated the influence of the g.66493737C/T SNP on speed indexes measured in a cohort of n = 85 Thoroughbred horses-in-training. We found significant associations between genotypes at the g.66493737C/T SNP and all measured speed variables: Dist(6) [distance travelled during 6 s before and after maximal velocity (V(max)); P = 0.0040], V(maxt) (duration at V(max); P = 0.0249), V(max) (P = 0.0265), Dist(6b) (distance travelled during 6 s before V(max); P = 0.0032), and Dist(6a)(distance travelled during 6 s after V(max); P = 0.0317). For each measure, horses with the C/C and C/T genotypes outperformed T/T horses, indicating the requirement for at least one C-allele to improve speed. For the most significantly associated variables (Dist(6) and Dist(6b)) the C/C cohort performed better than the T/T cohort with the heterozygotes intermediate, indicating a dose-dependent manifestation. These findings clearly indicate that variation at the MSTN gene influences speed in Thoroughbred horses.

Characterization of the responses of equine digital veins and arteries to calcitonin gene-related peptide.

OBJECTIVE: To compare responses of equine digital arteries (EDAs) and veins (EDVs) to human-αcalcitonin gene-related peptide (hαCGRP), evaluate effect of the endothelium, and characterize receptors and sources of endogenous CGRP. SAMPLE: Palmar digital vessels (5 to 9/experiment) from healthy adult horses killed at an abattoir. PROCEDURES: Vessel rings were mounted under tension in organ baths containing Krebs-Henseleit solution at 30 °C, with relaxation responses examined in vessels preconstricted with a thromboxane-mimetic (3 × 10(-8)M). Responses of endothelium-intact (+e) and -denuded (-e) EDAs and EDVs to hαCGRP C10(-10) to 3 × 10(-7)M) were compared. Following incubation with an hαCGRP receptor antagonist (hαCGRP(8-37); 1 µM), responses of EDA(-e) and EDV(-e) to hαCGRP (10(-7)M) were obtained. Responses of endothelium-intact and -denuded arteries and veins to hαCGRP (3 × 10(-7)M) or capsaicin (10(-5)M) were evaluated as well as responses of endothelium-intact and -denuded EDA and EDV to hαCGRP (10(-10) to 10(-6)M) after incubation with endothelin-1 (ET-1; 10(-12)M). RESULTS: hαCGRP resulted in nonendothelium, concentration-dependent relaxation in EDAs and EDVs, with greater responses in EDAs. Treatment with hαCGRP(8-37) had minimal effect on responses to hαCGRP in either vessel type. Capsaicin induced relaxation in both vessel types. There were no differences between responses to hαCGRP for vessels pretreated with ET-1 or vehicle. CONCLUSIONS AND CLINICAL RELEVANCE: Both hαCGRP and capsaicin induced digital vasodilation unaffected by a functional endothelium. This suggested that endogenous CGRP likely emanates from sensory-motor nerves and may contribute to digital vasodilation.

Circadian regulation of locomotor activity and skeletal muscle gene expression in the horse.

Circadian rhythms are innate 24-h cycles in behavioral and biochemical processes that permit physiological anticipation of daily environmental changes. Elucidating the relationship between activity rhythms and circadian patterns of gene expression may contribute to improved human and equine athletic performance. Six healthy, untrained mares were studied to determine whether locomotor activity behavior and skeletal muscle gene expression reflect endogenous circadian regulation. Activity was recorded for three consecutive 48-h periods: as a group at pasture (P), and individually stabled under a light-dark (LD) cycle and in constant darkness (DD). Halter-mounted Actiwatch-L data-loggers recorded light exposure and motor activity. Analysis of mean activity (average counts/min, activity bouts/day, average bout length) and cosinor parameters (acrophase, amplitude, mesor, goodness of fit) revealed a predominantly ultradian (8.9 ± 0.7 bouts/24 h) and weakly circadian pattern of activity in all three conditions (P, LD, DD). A more robust circadian pattern was observed during LD and DD. Muscle biopsies were obtained from the middle gluteal muscles every 4 h for 24 h under DD. One-way qRT-PCR results confirmed the circadian expression (P < 0.05) of six core clock genes (Arntl, Per1, Per2, Nr1d1, Nr1d2, Dbp) and the muscle-specific transcript, Myf6. Additional genes, Ucp3, Nrip1, and Vegfa, demonstrated P values approaching significance. These findings demonstrate circadian regulation of muscle function and imply that human management regimes may strengthen, or unmask, equine circadian behavioral outputs. As exercise synchronizes circadian rhythms, our findings provide a basis for future work determining peak times for training and competing horses, to reduce injury and to achieve optimal performance.

Characterization of the equine skeletal muscle transcriptome identifies novel functional responses to exercise training.

BACKGROUND: Digital gene expression profiling was used to characterize the assembly of genes expressed in equine skeletal muscle and to identify the subset of genes that were differentially expressed following a ten-month period of exercise training. The study cohort comprised seven Thoroughbred racehorses from a single training yard. Skeletal muscle biopsies were collected at rest from the gluteus medius at two time points: T(1) - untrained, (9 +/- 0.5 months old) and T(2) - trained (20 +/- 0.7 months old). RESULTS: The most abundant mRNA transcripts in the muscle transcriptome were those involved in muscle contraction, aerobic respiration and mitochondrial function. A previously unreported over-representation of genes related to RNA processing, the stress response and proteolysis was observed. Following training 92 tags were differentially expressed of which 74 were annotated. Sixteen genes showed increased expression, including the mitochondrial genes ACADVL, MRPS21 and SLC25A29 encoded by the nuclear genome. Among the 58 genes with decreased expression, MSTN, a negative regulator of muscle growth, had the greatest decrease.Functional analysis of all expressed genes using FatiScan revealed an asymmetric distribution of 482 Gene Ontology (GO) groups and 18 KEGG pathways. Functional groups displaying highly significant (P < 0.0001) increased expression included mitochondrion, oxidative phosphorylation and fatty acid metabolism while functional groups with decreased expression were mainly associated with structural genes and included the sarcoplasm, laminin complex and cytoskeleton. CONCLUSION: Exercise training in Thoroughbred racehorses results in coordinate changes in the gene expression of functional groups of genes related to metabolism, oxidative phosphorylation and muscle structure.

A sequence polymorphism in MSTN predicts sprinting ability and racing stamina in thoroughbred horses.

Variants of the MSTN gene encoding myostatin are associated with muscle hypertrophy phenotypes in a range of mammalian species, most notably cattle, dogs, mice, and humans. Using a sample of registered Thoroughbred horses (n = 148), we have identified a novel MSTN sequence polymorphism that is strongly associated (g.66493737C>T, P = 4.85x10(-8)) with best race distance among elite racehorses (n = 79). This observation was independently validated (P = 1.91x10(-6)) in a resampled group of Thoroughbreds (n = 62) and in a cohort of Thoroughbreds (n = 37, P = 0.0047) produced by the same trainer. We observed that C/C horses are suited to fast, short-distance races; C/T horses compete favorably in middle-distance races; and T/T horses have greater stamina. Evaluation of retrospective racecourse performance (n = 142) and stallion progeny performance predict that C/C and C/T horses are more likely to be successful two-year-old racehorses than T/T animals. Here we describe for the first time the identification of a gene variant in Thoroughbred racehorses that is predictive of genetic potential for an athletic phenotype.

Alterations in oxidative gene expression in equine skeletal muscle following exercise and training.

Intense selection for elite racing performance in the Thoroughbred horse (Equus caballus) has resulted in a number of adaptive physiological phenotypes relevant to exercise; however, the underlying molecular mechanisms responsible for these characteristics are not well understood. Adaptive changes in mRNA expression in equine skeletal muscle were investigated by real-time qRT-PCR for a panel of candidate exercise-response genes following a standardized incremental-step treadmill exercise test in eight untrained Thoroughbred horses. Biopsy samples were obtained from the gluteus medius before, immediately after, and 4 h after exercise. Significant (P < 0.05) differences in gene expression were detected for six genes (CKM, COX4I1, COX4I2, PDK4, PPARGC1A, and SLC2A4) 4 h after exercise. Investigation of relationships between mRNA and velocity at maximum heart rate (VHR(max)) and peak postexercise plasma lactate concentration ([La]T(1)) revealed significant (P < 0.05) associations with postexercise COX4I1 and PPARCG1A expression and between [La]T(1) and basal COX4I1 expression. Gene expression changes were investigated in a second cohort of horses after a 10 mo period of training. In resting samples, COX4I1 gene expression had significantly increased following training, and, after exercise, significant differences were identified for COX4I2, PDK4, and PPARGC1A. Significant relationships with VHR(max) and [La]T(1) were detected for PPARGC1A and COX4I1. These data highlight the roles of genes responsible for the regulation of oxygen-dependent metabolism, glucose metabolism, and fatty acid utilization in equine skeletal muscle adaptation to exercise.

A genome scan for positive selection in thoroughbred horses.

Thoroughbred horses have been selected for exceptional racing performance resulting in system-wide structural and functional adaptations contributing to elite athletic phenotypes. Because selection has been recent and intense in a closed population that stems from a small number of founder animals Thoroughbreds represent a unique population within which to identify genomic contributions to exercise-related traits. Employing a population genetics-based hitchhiking mapping approach we performed a genome scan using 394 autosomal and X chromosome microsatellite loci and identified positively selected loci in the extreme tail-ends of the empirical distributions for (1) deviations from expected heterozygosity (Ewens-Watterson test) in Thoroughbred (n = 112) and (2) global differentiation among four geographically diverse horse populations (F(ST)). We found positively selected genomic regions in Thoroughbred enriched for phosphoinositide-mediated signalling (3.2-fold enrichment; P<0.01), insulin receptor signalling (5.0-fold enrichment; P<0.01) and lipid transport (2.2-fold enrichment; P<0.05) genes. We found a significant overrepresentation of sarcoglycan complex (11.1-fold enrichment; P<0.05) and focal adhesion pathway (1.9-fold enrichment; P<0.01) genes highlighting the role for muscle strength and integrity in the Thoroughbred athletic phenotype. We report for the first time candidate athletic-performance genes within regions targeted by selection in Thoroughbred horses that are principally responsible for fatty acid oxidation, increased insulin sensitivity and muscle strength: ACSS1 (acyl-CoA synthetase short-chain family member 1), ACTA1 (actin, alpha 1, skeletal muscle), ACTN2 (actinin, alpha 2), ADHFE1 (alcohol dehydrogenase, iron containing, 1), MTFR1 (mitochondrial fission regulator 1), PDK4 (pyruvate dehydrogenase kinase, isozyme 4) and TNC (tenascin C). Understanding the genetic basis for exercise adaptation will be crucial for the identification of genes within the complex molecular networks underlying obesity and its consequential pathologies, such as type 2 diabetes. Therefore, we propose Thoroughbred as a novel in vivo large animal model for understanding molecular protection against metabolic disease.

Transcriptional adaptations following exercise in thoroughbred horse skeletal muscle highlights molecular mechanisms that lead to muscle hypertrophy.

BACKGROUND: Selection for exercise-adapted phenotypes in the Thoroughbred racehorse has provided a valuable model system to understand molecular responses to exercise in skeletal muscle. Exercise stimulates immediate early molecular responses as well as delayed responses during recovery, resulting in a return to homeostasis and enabling long term adaptation. Global mRNA expression during the immediate-response period has not previously been reported in skeletal muscle following exercise in any species. Also, global gene expression changes in equine skeletal muscle following exercise have not been reported. Therefore, to identify novel genes and key regulatory pathways responsible for exercise adaptation we have used equine-specific cDNA microarrays to examine global mRNA expression in skeletal muscle from a cohort of Thoroughbred horses (n = 8) at three time points (before exercise, immediately post-exercise, and four hours post-exercise) following a single bout of treadmill exercise. RESULTS: Skeletal muscle biopsies were taken from the gluteus medius before (T(0)), immediately after (T(1)) and four hours after (T(2)) exercise. Statistically significant differences in mRNA abundance between time points (T(0) vs T(1) and T(0) vs T(2)) were determined using the empirical Bayes moderated t-test in the Bioconductor package Linear Models for Microarray Data (LIMMA) and the expression of a select panel of genes was validated using real time quantitative reverse transcription PCR (qRT-PCR). While only two genes had increased expression at T(1) (P < 0.05), by T(2) 932 genes had increased (P < 0.05) and 562 genes had decreased expression (P < 0.05). Functional analysis of genes differentially expressed during the recovery phase (T(2)) revealed an over-representation of genes localized to the actin cytoskeleton and with functions in the MAPK signalling, focal adhesion, insulin signalling, mTOR signaling, p53 signaling and Type II diabetes mellitus pathways. At T(1), using a less stringent statistical approach, we observed an over-representation of genes involved in the stress response, metabolism and intracellular signaling. These findings suggest that protein synthesis, mechanosensation and muscle remodeling contribute to skeletal muscle adaptation towards improved integrity and hypertrophy. CONCLUSIONS: This is the first study to characterize global mRNA expression profiles in equine skeletal muscle using an equine-specific microarray platform. Here we reveal novel genes and mechanisms that are temporally expressed following exercise providing new knowledge about the early and late molecular responses to exercise in the equine skeletal muscle transcriptome.

Characterization and comparison of the responses of equine digital arteries and veins to endothelin-1.

OBJECTIVE: To compare the responses of equine digital arteries (EDAs) and equine digital veins (EDVs) to endothelin-1 (ET-1) and determine the role of the endothelium and type of receptors involved in the modulation and mediation of those responses, respectively. SAMPLE POPULATION: 5 to 9 palmar digital vessels/experiment from 28 healthy horses. PROCEDURE: Rings of dissected vessels were mounted under tension between force transducer wires in organ baths containing Krebs-Henseleit solution at 30 degrees C. Responses of EDAs and EDVs (with intact [+e] or denuded [-e] endothelium) to cumulative concentrations of ET-1 (10(-10) to 3 X 10(-7) M) were compared. For (+e)EDAs and (+e)EDVs precontracted with a thromboxane-mimetic (U44069; 10(-8) M) and (-e)EDAs and (-e)EDVs, responses to an ETB receptor agonist (S6c; 10(-10) to 3 X 10(-7) M) were evaluated. Responses to ET-1 (10(-7) M) in (-e)EDAs and (-e)EDVs were evaluated after incubation with an ETA receptor antagonist (BQ-123; 3 X 10(-7) M), an ETB receptor antagonist (BQ-788; 3 X 10(-7) M), or vehicle solution. RESULTS: Endothelin-1 induced a concentration-dependent contraction of endothelium-intact and -denuded EDAs and EDVs; EDVs were more sensitive. Neither vessel type relaxed in response to S6c, although 2 of the (-e)EDAs contracted mildly. Whereas BQ-123 inhibited the (-e)EDA and (-e)EDV responses to ET-1, BQ-788 had no effect. CONCLUSIONS AND CLINICAL RELEVANCE: Endothelin-1 induced digital vasoconstriction (marked constriction in veins). This action was unaffected by endothelium and mediated predominantly by ETA receptors. These findings suggest ET-1 can induce selective digital venoconstriction.