Rita G. Fonseca

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 = 4.85×10−8) with best race distance among elite racehorses (n = 79). This observation was independently validated (P = 1.91×10−6) 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.

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

BackgroundDigital 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: T1 - untrained, (9 ± 0.5 months old) and T2 - trained (20 ± 0.7 months old).ResultsThe 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.ConclusionExercise 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.

mRNA expression of genes regulating oxidative phosphorylation in the muscle of beef cattle divergently ranked on residual feed intake

Our objective was to evaluate the effects of phenotypic ranking on residual feed intake (RFI) on the transcription of genes 1) involved in the respiratory chain complex and 2) coding for transcriptional factors regulating mitochondrial biogenesis, across two contrasting diet types. Beef heifers (n = 86) fed a diet comprising 70:30 concentrate-corn silage [low forage (LF)] over a 82-day period were ranked on RFI. The 10 highest (feed inefficient, high-RFI) and 10 lowest (feed efficient, low-RFI) ranking animals were selected for the current study. Biopsies of the M. longissimus dorsi were harvested following initial selection (LF diet) and again following a 6 wk period while the animals were offered a high-forage (HF) grass silage-only diet. Real-time PCR was used to quantify mRNA transcripts of 17 genes associated with cellular energetic efficiency. The mRNA expression of UCP3 tended to be upregulated (2.2-fold, P = 0.06) for the high-RFI compared with the low-RFI animals. mRNA transcripts coding for the transcription factor PGC-1α was 1.7-fold higher (P = 0.01) in low compared with high-RFI animals. A phenotype × diet interaction was evident for the abundance of ANT1 mRNA transcript, with greater (P = 0.04) expression levels detected in the low-RFI phenotype during the HF period, but no difference (P = 0.50) between phenotypes during the LF period. A phenotype × diet interaction was also evident for COX II with greater expression levels detected (P = 0.04) in the low compared with the high RFI phenotype while on LF but not the HF diet (P = 0.22). These data suggest an association between cellular energetic efficiency and RFI in cattle.

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.

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-β 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-β 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.

The association of various speed indices to training responses in Thoroughbred flat racehorses measured with a global positioning and heart rate monitoring system

REASONS FOR PERFORMING STUDY Fitness assessment can be challenging. The use of global positioning systems (GPS) with heart rate (HR) monitors has been promising; however, evaluation of speed parameters during training has not been reported. OBJECTIVES To evaluate speed indices during training in Thoroughbreds using a GPS-HR monitor. METHODS Thoroughbreds (n = 102) were assessed during training with data collected each work day (WD; sprinting). Speed indices evaluated included maximal velocity (V(max)), duration at V(max) (V(maxt)), acceleration rate (m/s(2)) from 800 m to V(max) (Acc800-V(max)), the distance (m) 6 (V(maxD6)) and 12 (V(maxD12)) s before (acceleration [a]) and after (deceleration [d]) V(max) and the deceleration rate from V(max) to the finish (V(maxDFd)). Blood for plasma lactate ([LA]) and creatine kinase ([CK]) measurements were taken before (T(0)), 5 mins (T(1)) and 6 h after exercise (T(2)). WD accumulation, jockey, gallop condition, horse gender, age, total distance covered (DistT), maximum HR (HR(max)), velocity at 200 beats/min (V(200)) and velocity at maximum HR (VHR(max)) for each WD were evaluated for associations with [LA], [CK], speed indices and racing performance. Data were analysed by repeated measures ANOVA with P < 0.05 significant. RESULTS No speed parameter clearly changed with training. Gallop condition affected V(max), V(maxt) and all distances covered with V(max) and distances increasing and V(maxt) decreasing as gallop surface became firmer. Jockey influenced V(max), V(maxD6a) and all decelerations, while DistT was inversely associated with Acc800-V(max), HR(max) and V(200) and positively associated with V(max), all accelerations and decelerations. [LA] at T(1) was positively associated with DistT and V(maxDFd). CONCLUSIONS Speed parameters did not change with training but were affected by jockey, gallop condition and exercise distance. This information may help to modify training to maximise fitness, minimise injury and choose distances best suited for individuals.

The relationship between body composition, training and race performance in a group of Thoroughbred flat racehorses

REASONS FOR PERFORMING STUDY Few noninvasive measures associated with performance assessment are available for racehorse trainers. Evaluation of body composition of superior human sprinters has revealed a lower fat mass (FM), percentage (%) fat and greater fat-free mass (FFM), but to date there have been few studies evaluating this in racehorses. OBJECTIVES To determine the effects of age, gender and training on body composition and the relationship between body composition, physiological measurements and performance in Thoroughbred racehorses. METHODS At 2, 5 and 8 months of training, rump fat thickness (RFT) was ultrasonographically measured in 1-, 2- and 3-year-old Thoroughbreds (n = 148), with FM, % fat and FFM calculated. Speed, heart rate, plasma lactate and serum creatine kinase concentrations were recorded during each fast work session. Training duration (number of training days) and intensity (number of fast work sessions) were collated for each training period. Retrospective racing performance was used to categorise horses as elite or nonelite. RESULTS FFM was greater in males (P = 0.006) at all training stages. There were no interactions between training duration, intensity, gender and age (P>0.05); all effects were linear. Training duration had a negative effect on RFT (P = 0.0002), FM (P<0.0001) and % fat (P<0.0001) and a positive effect on FFM (P = 0.01). Training intensity had a negative effect on RFT (P = 0.009), FM (P<0.0001), % fat (P<0.0001) and FFM (P<0.0001). FFM was greater for elite vs. nonelite horses at all training stages (P = 0.003), for males (P = 0.05) and females (P = 0.04) and for 2- (P = 0.002) and 3-year-olds (P = 0.02). CONCLUSIONS While age and training affect body composition, FFM is associated with performance. POTENTIAL RELEVANCE Body composition assessment may assist fitness and performance evaluation.

Effect of training on plasma myeloperoxidase concentrations measured before and following intense exercise in Thoroughbred racehorses

Exercise in horses induces neutrophil degranulation and subsequent increases in plasma myeloperoxidase concentrations (MPO). It is not known whether this response is affected by training or the sampling time in relation to exercise. Our objective was to evaluate plasma MPO concentration at different time points in response to exercise in Thoroughbreds before and following high-intensity training and to evaluate relationships between plasma MPO concentration, physiological measurements and performance. Throughbred racehorses in active training and racing (n=26) performed an exercise test on a high-speed treadmill at least once at the beginning (first three months), middle (second three months) or end (last three months) of a nine month training period with training intensity (number of fast work sessions) collated for each period. Heart rate, speed and distance were recorded and venous blood collected before (T0), during and up to maximal speed (TVmax), 5 min (T5min) and 4 h (T4h) following exercise for me...