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Summary: Fractures are medical conditions that compromise the athletic potential of horses and/or the safety of jockeys. Therefore, the reduction of fracture risk is an important horse and human welfare issue. The present study used molecular genetic approaches to determine the effect of genetic risk for fracture at four candidate SNPs spanning the myostatin (MSTN) gene on horse chromosome 18. Among the 3706 Japanese Thoroughbred racehorses, 1089 (29.4%) had experienced fractures in their athletic life, indicating the common occurrence of this injury in Thoroughbreds. In the case/control association study, fractures of the carpus (carpal bones and distal radius) were statistically associated with g.65809482T/C (P = 1.17 x 10‐8), g.65868604G/T (P = 2.66 x 10‐9), and g.66493737C/T (P = 6.41 x 10‐8). In the retrospective cohort study using 1710 racehorses born in 2000, the relative risk (RR) was highest for male horses at g.65868604G/T, based on the dominant allele risk model (RR = 2.251, 95% confidence interval 1.407–3.604, P = 0.00041), and for female horses at g.65868604G/T, based on the recessive allele risk model (RR = 2.313, 95% confidence interval 1.380–3.877, P = 0.00163). Considering the association of these SNPs with racing performance traits such as speed, these genotypes may affect the occurrence of carpus fractures in Japanese Thoroughbred racehorses as a consequence of the non‐genetic influence of the genotype on the distance and/or intensity of racing and training. The genetic information presented here may contribute to the development of strategic training programs and racing plans for racehorses that improve their health and welfare. [ABSTRACT FROM AUTHOR]

Summary: Despite strong selection for athletic traits in Thoroughbred horses, there is marked variation in speed and aptitude for racing performance within the breed. Using global positioning system monitoring during exercise training, we measured speed variables and temporal changes in speed with age to derive phenotypes for GWAS. The aim of the study was to test the hypothesis that genetic variation contributes to variation in end‐point physiological traits, in this case galloping speed measured during field exercise tests. Standardisation of field‐measured phenotypes was attempted by assessing horses exercised on the same gallop track and managed under similar conditions by a single trainer. PCA of six key speed indices captured 73.9% of the variation with principal component 1 (PC1). Verifying the utility of the phenotype, we observed that PC1 (median) in 2‐year‐old horses was significantly different among elite, non‐elite and unraced horses (P < 0.001) and the temporal change with age in PC1 varied among horses with different myostatin (MSTN) g.66493737C>T SNP genotypes. A GWAS for PC1 in 2‐year‐old horses (n = 122) identified four SNPs reaching the suggestive threshold for association (P < 4.80 × 10−5), defining a 1.09 Mb candidate region on ECA8 containing the myosin XVIIIB (MYO18B) gene. In a GWAS for temporal change in PC1 with age (n = 168), five SNPs reached the suggestive threshold for association and defined candidate regions on ECA2 and ECA11. Both regions contained genes that are significantly differentially expressed in equine skeletal muscle in response to acute exercise and training stimuli, including MYO18A. As MYO18A plays a regulatory role in the skeletal muscle response to exercise, the identified genomic variation proximal to the myosin family genes may be important for the regulation of the response to exercise and training. [ABSTRACT FROM AUTHOR]

Summary: Background: Race distance aptitude in Thoroughbred horses is highly heritable and is influenced largely by variation at the myostatin gene (MSTN). Objectives: In addition to MSTN, we hypothesised that other modifying loci contribute to best race distance. Study design: Using 3006 Thoroughbreds, including 835 'elite' horses, which were >3 years old, had race records and were sampled from Europe/Middle‐East, Australia/New Zealand, North America and South Africa, we performed genome‐wide association (GWA) tests and separately developed a genomic prediction algorithm to comprehensively catalogue additive genetic variation contributing to best race distance. Methods: 48,896 single‐nucleotide polymorphism (SNP) genotypes were generated from high‐density SNP genotyping arrays. Heritability estimates, tests of GWA and genomic prediction models were derived for the phenotypes: average race distance, best race distance for elite, nonelite and all winning horses. Results: Heritability estimates were high (hm2 = 0.51, best race distance – elite; hm2 = 0.42, best race distance – nonelite; hm2 = 0.40, best race distance – all) and most of the variation was attributed to the MSTN gene. MSTN locus SNPs were the most strongly associated with the trait and included BIEC2‐438999 (ECA18:66913090; P = 4.51 × 10−110, average race distance; P = 2.33 × 10−42, best race distance – elite). The genomic prediction algorithm enabled the inclusion of variation from all SNPs in a model that partitioned horses into short and long cohorts following assignment of MSTN genotype. Additional genes with minor contributions to best race distance were identified. Main limitations: The nongenetic influence of owner/trainer decisions on placement of horses in suitable races could not be controlled. Conclusions: MSTN is the single most important genetic contributor to best race distance in the Thoroughbred. Employment of genetic prediction models will lead to more accurate placing of horses in races that are best suited to their inherited genetic potential for distance aptitude. [ABSTRACT FROM AUTHOR]

Summary: Background: We hypothesised that grade/appearance for upper respiratory tract (URT) disorders identified at the first overground endoscopy (OGE) examination would vary at subsequent examinations. Objectives: To compare OGE examinations from horses evaluated on at least two occasions under similar exercise conditions without treatment intervention. Study design: Retrospective cohort. Methods: Pre‐exercise and exercising OGE recordings from Thoroughbred horses undergoing multiple examinations under similar exercise conditions were reviewed, with the first two recordings for each horse statistically evaluated. Paired Wilcoxon signed‐rank tests were used to assess differences in exercise and physiological parameters between examinations. Z‐tests were used to assess the proportion of changes in URT disorder grade/appearance between examinations. A McNemar's test was used to compare the proportion of horses with each disorder at each examination. Test–retest reliability across examinations was assessed using Spearman's ρ, and ordered logistic regression used to explore temporal effects on repeatability. Lattice plots were constructed to view variability in disorders over time. Results: Seventy‐eight horses (median age 2.4 years) with 195 resting endoscopic examinations including 72/78 horses with 179 pre‐exercise and exercising OGE examinations were evaluated. Median time between examinations was 226.5 days with no differences between exercise and physiological parameters. Grades significantly varied between examinations for all disorders, and in particular for palatal instability (PI) and epiglottic grade at rest. A temporal link between examination interval and disorder grade change was identified for PI and resting arytenoid asymmetry. Main limitations: OGE re‐examinations within a shorter, more consistent time‐frame would allow determination of intrahorse variability. The sample size was inadequate to conclusively establish temporal links between disorders and time between examinations. Conclusions: Variability for most URT abnormalities identified with OGE should be considered when making therapeutic decisions based on a single examination and may partially explain development of additional URT conditions after surgical intervention. The Summary is available in Portuguese ‐ see Supporting Information [ABSTRACT FROM AUTHOR]

Summary: Background: Endoscopic tip placement in the pharynx and water flushing interval (FI) may affect exercising upper respiratory tract (URT) endoscopic results. Objectives: To determine associations between the endoscopic tip position in the pharynx and automated FI with overground endoscopic (OGE) results. Study design: Randomised balanced 2X5 factorial design. Methods: A total of n = 200 horses undergoing OGE were randomly assigned into 10 groups (n = 20/group) of different automated endoscopic FIs (no flushing, 60, 120, 180, 240 s) with the endoscope tip positioned either rostrally (position A) or caudally (position B) in the pharynx. Endoscopic videos were analysed and all URT abnormalities graded using published scales. Disorders with ≤10% prevalence were excluded from the final analysis with only arytenoid asymmetry at exercise (AAex), vocal fold collapse (VFC), palatal dysfunction (PD) and medial deviation of the aryepiglottic folds (MDAF) included. The association of endoscope position and FI with URT disorders was assessed using ordinal regression models with P≤0.05 significant. Results: Endoscope tip positioning was significantly associated with PD grading (P = 0.002), with 63/100 horses diagnosed with PD in position A and 45/100 in position B. No other significant direct associations between URT disease and endoscope tip position were identified, although interactions between exercise velocity and endoscope position affected MDAF grade. FI was not directly associated with alterations in disorder grading, although interactions between exercise velocity and FI appeared to affect MDAF grade. Main limitations: The same horse was not evaluated under each test condition potentially resulting in sample bias. Interactions between disorders were not evaluated. The sample size was insufficient to conclusively explore relationships between all factors and disorder grading. Conclusions: Position of the endoscope tip within the pharynx appears to affect grading of PD during OGE examination. Exercise velocity may affect MDAF grade through interactions with endoscope position and FI. [ABSTRACT FROM AUTHOR]

Summary: Background: Conflicting results have been reported for risk factors for recurrent laryngeal neuropathy (RLN) based on resting endoscopic evaluation and comparison of single conformation traits, with many traits correlated to one another. Objectives: To simplify identification of signalment and conformation traits (i.e. variables) associated with RLN cases and controls diagnosed with exercising overground endoscopy (OGE) using exploratory factor analysis (EFA). Study design: Prospective cohort. Methods: Pearson's rank correlation was used to establish significance and association between variables collected from n = 188 Thoroughbreds from one stable by observers blinded to OGE results. Exploratory factor analysis was conducted on nine variables for cases and controls; common elements between variables developed a factor, with variables grouped into three factors for cases and controls respectively. Correlation (loading) between each variable and factor was calculated to rank relationships between variables and cases/controls, with factors retrospectively named based on their underlying correlations with variables. Results: Numerous inter‐correlations were present between variables. Most strongly correlated in cases were wither height with body weight (r = 0.70) and ventral neck length (r = 0.68) and in controls body weight with rostral neck circumference (r = 0.58). Wither height (r = 0.61) significantly loaded the top‐ranked factor for cases ('heightRLN'), explaining 25% of conformational variance. Ventral neck length (r = 0.69) and age (r = 0.57) significantly loaded the second‐ranked factor for cases ('neck lengthRLN'), explaining 16% of conformational variance. Rostral neck circumference (r = 0.86) and body weight (r = 0.6) significantly loaded the top‐ranked factor for controls ('body sizeCON'), explaining 19% of the variance. Wither height (r = 0.84) significantly loaded the second‐ranked factor for controls ('heightCON'), explaining 13% of the variance. Main limitations: Horses had not reached skeletal maturity. Conclusions: Exploratory factor analysis allowed weightings to be determined for each variable. Wither height was the predominant conformational feature associated with RLN. Exploratory factor analysis confirms aggregated conformational differences exist between RLN cases and controls, suitable for future evaluations. [ABSTRACT FROM AUTHOR]

Summary: Adaptation to early training and racing (i.e. precocity), which is highly variable in racing Thoroughbreds, has implications for the selection and training of horses. We hypothesised that precocity in Thoroughbred racehorses is heritable. Age at first sprint training session (work day), age at first race and age at best race were used as phenotypes to quantify precocity. Using high‐density SNP array data, additive SNP heritability ( h SNP 2) was estimated to be 0.17, 0.14 and 0.17 for the three traits respectively. In genome‐wide association studies (GWAS) for age at first race and age at best race, a 1.98‐Mb region on equine chromosome 18 (ECA18) was identified. The most significant association was with the myostatin (MSTN) g.66493737C>T SNP (P = 5.46 × 10−12 and P = 1.89 × 10−14 respectively). In addition, two SNPs on ECA1 (g.37770220G>A and g.37770305T>C) within the first intron of the serotonin receptor gene HTR7 were significantly associated with age at first race and age at best race. Although no significant associations were identified for age at first work day, the MSTN:g.66493737C>T SNP was among the top 20 SNPs in the GWAS (P = 3.98 × 10−5). Here we have identified variants with potential roles in early adaptation to training. Although there was an overlap in genes associated with precocity and distance aptitude (i.e. MSTN), the HTR7 variants were more strongly associated with precocity than with distance. Because HTR7 is closely related to the HTR1A gene, previously implicated in tractability in young Thoroughbreds, this suggests that behavioural traits may influence precocity. [ABSTRACT FROM AUTHOR]

Background: Circulating miRNAs (ci-miRNAs) are endogenous, non-coding RNAs emerging as potential diagnostic biomarkers. Equine miRNAs have been previously identified including subsets of tissue-specific miRNAs. In order to investigate ci-miRNAs as diagnostic tools, normal patterns of expression for different scenarios including responses to exercise need to be identified. Human studies have demonstrated that many ci-miRNAs are up-regulated following exercise with changes in expression patterns in skeletal muscle. However, technical challenges such as haemolysis impact on accurate plasma ci-miRNA quantification, with haemolysis often occurring naturally in horses following moderateto- intense exercise. The objectives of this study were to identify plasma ci-miRNA profiles and skeletal muscle miRNAs before and after exercise in Thoroughbreds (Tb), and to evaluate for the presence and effect of haemolysis on plasma ci-miRNA determination. Resting and post-exercise plasma ci-miRNA profiles and haemolysis were evaluated in twenty 3 year-old Tbs in sprint training. Resting and post-exercise skeletal muscle miRNA abundance was evaluated in a second cohort of eleven 2 year-old Tbs just entering sprint training. Haemolysis was further quantified in resting blood samples from twelve Tbs in sprint training. A human plasma panel containing 179 miRNAs was used for profiling, with haemolysis assessed spectrophotometrically. Data was analysed using a paired Student's t-test and Pearson's rank correlation. Results: Plasma ci-miRNA data for 13/20 horses and all skeletal muscle miRNA data passed quality control. From plasma, 52/179 miRNAs were detected at both time-points. Haemolysis levels were greater than the threshold for accurate quantification of ci-miRNAs in 18/25 resting and all post-exercise plasma samples. Positive correlations (P < 0.05) between haemolysis and miRNA abundance were detected for all but 4 miRNAs, so exercise-induced changes in plasma ci-miRNA expression could not be quantified. In skeletal muscle samples, 97/179 miRNAs were detected with 5 miRNAs (miR-21-5p, let-7d-3p, let-7d-5p, miR-30b-5p, miR-30e-5p) differentially expressed (DE, P < 0.05) between time-points. Conclusions: The degree of haemolysis needs to be determined prior to quantifying plasma ci-miRNA expression from horses in high-intensity exercise training. Identification of DE miRNAs in skeletal muscle indicates modification of miRNA expression may contribute to adaptive training responses in Tbs. Using a human plasma panel likely limited detection of equine-specific miRNAs. [ABSTRACT FROM AUTHOR]

Background Previous studies support good intra- and interobserver agreements for endoscopic evaluation of various upper respiratory tract ( URT) diseases in horses. However, these studies mainly assessed resting endoscopic examination videos and/or focussed on a single URT abnormality. Objectives To estimate intra- and interobserver agreement for identification and grading of all URT abnormalities from resting and overground endoscopy ( OGE) videos of Thoroughbreds. Study design Blinded, fully crossed design. Methods Resting and OGE URT videos for n = 43 Thoroughbreds were retrospectively chosen based on identification of common URT disorders. The videos were randomly evaluated in duplicate by 4 raters blinded to all information including prior URT disorder(s) diagnosis. Abnormalities were graded using well-described ordinal scales. Intra- and interobserver agreements were estimated using Cohen's weighted κ and Krippendorff's α, respectively. Results Intraobserver agreement was perfect/nearly perfect for arytenoid symmetry at exercise, epiglottic entrapment and epiglottic retroversion, substantial for arytenoid asymmetry at rest, palatal dysfunction ( PD), medial deviation of the aryepiglottic folds (MDAF), pharyngeal mucus and epiglottic grade at exercise and moderate for vocal fold collapse ( VFC), ventromedial luxation of the apex of the corniculate process of the arytenoid (VLAC), nasopharyngeal collapse ( NPC) and epiglottic grade at rest. Interobserver agreement was substantial for arytenoid symmetry at exercise and PD and moderate for arytenoid asymmetry at rest, MDAF, VLAC and epiglottic entrapment. It was only fair for VFC, epiglottic grade at exercise, epiglottic retroversion, pharyngeal mucus and NPC and poor for epiglottic grade at rest. Main limitations Sample size was insufficient to allow assessment of the effect of one abnormality on the grading of another abnormality. Conclusions Observers were consistent in grading URT disorders. However, significant disparity in grading existed between observers for some conditions affecting reliability. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The recent completion of the horse genome and commercial availability of an equine SNP genotyping array has facilitated the mapping of disease genes. We report putative localization of the gene responsible for dwarfism, a trait in Friesian horses that is thought to have a recessive mode of inheritance, to a 2- MB region of chromosome 14 using just 10 affected animals and 10 controls. We successfully genotyped 34 429 SNPs that were tested for association with dwarfism using chi-square tests. The most significant SNP in our study, BIEC2-239376 ( P = 4.54 × 10, P = 7.74 × 10), is located close to a gene implicated in human dwarfism. Fine-mapping and resequencing analyses did not aid in further localization of the causative variant, and replication of our findings in independent sample sets will be necessary to confirm these results. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA (mtDNA) D-loop sequences (381 bp) from 100 thoroughbreds in 19 of the most common matrilineal female families were used to reconstruct a founder female population for the thoroughbred (∼1650–1750 AD). Seventeen haplotypes were found to have contributed to the 19 female lineages. In order to place the reconstructed founder population in wider historical context, we examined, using both single strand conformation polymorphism and direct sequence analysis, variation in a 343 bp mtDNA fragment in that population and 13 other horse populations of disparate provenance. Interpopulation diversity analyses revealed no significant difference in variation between the thoroughbred founder population and the 13 other diverse horse populations and suggested a non-random partitioning of diversity among geographically diverse horse populations. Within thoroughbreds, almost half of the female families, which are each considered from pedigrees to have matrilines converging to one of >30 historically recognized female ancestors, contained sequences which were inconsistent with common descent. Examination of the anomalies in the context of pedigree records suggests the majority might be best explained as confusion of mares at the foundation stages, although some could have some bearing on more recent (19th century – 1980) lineages. We have used this combination of molecular and historical information to identify some of the founder dams and to make new interpretations about the early history of the thoroughbred. [ABSTRACT FROM AUTHOR]