Comparison of ventilatory and oxygen consumption measurements of yearling Thoroughbred colts and fillies exercising unridden on an all-weather track.

Sex effects on ventilatory and oxygen consumption (V̇O2) measurements during exercise have been identified in humans. This study's aim was to evaluate the hypothesis that there are sex effects on ventilatory and V̇O2 measurements in exercising, untrained yearling Thoroughbreds (Tb). Forty-one Tbs (16 colts, 25 fillies; 19.8 ± 1.4 months old) were recruited. Physiological, ventilatory and exercise data were gathered from horses exercising unridden at high intensity on an all-weather track from a global positioning-heart rate unit and a portable ergospirometry system. Data were analysed with an unpaired Student's t-test and the Benjamini-Hochberg correction for multiple testing (P ≤ 0.05 significant). Mean bodyweight (BW, P = 0.002) and wither height (P = 0.04) were greater for colts than fillies. There were no differences in physiological and exercise data and absolute peak V̇O2 between groups. However, fillies had a higher mass specific peak V̇O2 (P = 0.03) than colts (121.5 ± 21.6 mL/kg.min vs. 111.9 ± 27.4 mL/kg.min). The peak breathing frequency was greater for fillies (P < 0.001) while the peak inspiratory (P < 0.001) and expiratory air flow (P < 0.001), peak expiratory tidal volume (VTE; P < 0.001) and peak minute ventilation (V̇E; P = 0.01) were greater for colts; there were no differences for peak VTE and V̇E when adjusted for BW. Differences in BW explain the differences in mass specific peak V̇O2 between groups. Given their morphological differences, it is likely that lung volumes and airway diameters are smaller for fillies, resulting in greater resistance and lower air flows and volumes. Further research is required to investigate the ventilatory differences and how they may change with maturation and impact performance.

Analysis of genetic variation contributing to measured speed in Thoroughbreds identifies genomic regions involved in the transcriptional response to exercise.

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.

Impact of pharyngeal endoscopic tip placement and water flushing interval on upper respiratory tract disorders in horses undergoing overground endoscopy.

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.

Serial evaluation of resting and exercising overground endoscopic examination results in young Thoroughbreds with no treatment intervention.

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.

Exploratory factor analysis of signalment and conformational measurements in Thoroughbred horses with and without recurrent laryngeal neuropathy.

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.

Evaluation of microRNA expression in plasma and skeletal muscle of thoroughbred racehorses in training.

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 moderate-to-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.

Intra- and interobserver reliability estimates for identification and grading of upper respiratory tract abnormalities recorded in horses at rest and during overground endoscopy.

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.