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Evidence suggests that positive pacing strategy improves exercise performance and fatigue tolerance in athletic events lasting 1-5 min. This study investigated muscle metabolic responses to positive and negative pacing strategies in Thoroughbred horses. Eight Thoroughbred horses performed 2 min treadmill running using positive (1 min at 110% maximal O2 uptake [V̇O2max], followed by 1 min at 90% V̇O2max) and negative (1 min at 90% V̇O2max, followed by 1 min at 110% V̇O2max) pacing strategies. The arterial-mixed venous O2 difference did not significantly differ between the two strategies. Plasma lactate levels increased toward 2 min, with significantly higher concentrations during positive pacing than during negative pacing. Muscle glycogen level was significantly lower at 1 and 2 min of positive pacing than those of negative pacing. Metabolomic analysis showed that the sum of glycolytic intermediates increased during the first half of positive pacing and the second half of negative pacing. Regardless of pacing strategy, the sum of tricarboxylic acid cycle metabolites increased during the first half but remained unchanged thereafter. Our data suggest that positive pacing strategy is likely to activate glycolytic metabolism to a greater extent compared to negative pacing, even though the total workload is identical.

Equus caballus papillomavirus 2 (EcPV2) is known to cause genital neoplasms in horses. However, reports on EcPV2 in Japan and Asia are limited. Herein, we present the histopathological and virological findings of the first reported case of an EcPV2-associated penile mass in Japan. The patient was a 22-year-old stallion with a history of breeding in Japan and abroad. Histopathological examination contained RNA in situ hybridization targeting the E6/E7 region and an immunohistochemical approach, and whole-genome sequencing was conducted within the viral examination. Proliferating epidermal cells were observed, and EcPV2 E6/E7 mRNA was detected within the epidermis, which was interpreted as viral papilloma. The detected EcPV2 virus was genetically close to foreign strains and different from the strain previously reported from a Japanese mare. This suggests that various types of EcPV2 might already exist among horses in Japan. Although the mass reported herein was not malignant based on histopathological findings and the absence of recurrence, its presence on the penis would be an obstacle to breeding. These results provide a better understanding of the pathogenesis and diversity of EcPV2.

Information regarding the histopathology of the proximal phalanx (P1) sagittal groove in racehorses is limited. Twenty-nine cadaver limbs from nine Thoroughbred racehorses in racing/race-training underwent histological examination. Histological specimens of the third metacarpal/metatarsal (MC3/MT3) parasagittal grooves and P1 sagittal grooves were graded for histopathological findings in hyaline cartilage (HC), calcified cartilage (CC), and subchondral plate and trabecular bone (SCB/TB) regions. Histopathological grades were compared between (1) fissure and non-fissure locations observed in a previous study and (2) dorsal, middle, and palmar/plantar aspects. (1) HC, CC, and SCB/TB grades were more severe in fissure than non-fissure locations in the MC3/MT3 parasagittal groove (p < 0.001). SCB/TB grades were more severe in fissure than non-fissure locations in the P1 sagittal groove (p < 0.001). (2) HC, CC, and SCB/TB grades including SCB collapse were more severe in the palmar/plantar than the middle aspect of the MC3/MT3 parasagittal groove (p < 0.001). SCB/TB grades including SCB collapse were more severe in the dorsal and middle than the palmar/plantar aspect of the P1 sagittal groove (p < 0.001). Histopathology in the SCB/TB region including bone fatigue injury was related to fissure locations, the palmar/plantar MC3/MT3 parasagittal groove, and the dorsal P1 sagittal groove.

Gene doping is prohibited in horse sports and can involve the administration of exogenous genes, called transgenes, to postnatal animals. Quantitative polymerase chain reaction (qPCR) methods have been developed to detect gene doping; however, these generally require DNA extraction from the plasma prior to qPCR. In this study, we developed two methods, direct droplet digital PCR (ddPCR) and nested ddPCR, to detect the equine erythropoietin (EPO) transgene without DNA extraction. Direct ddPCR used pretreated plasma and PCR to detect the EPO transgene spiked at 10 copies/μL. Nested ddPCR utilised pre-amplification using nontreated plasma, purification of PCR products and PCR to detect the EPO transgene spiked at 1 copy/μL in plasma. These methods successfully detected the EPO transgene after intramuscular injection into horses. Since each method has different detection sensitivity, the combined use of direct ddPCR for screening and nested ddPCR for confirmation may complement each other and prevent the occurrence of false positives, allowing the reliable detection of gene-doped substances. One advantage of these methods is the small amount of sample required, approximately 2.2-5.0 μl, owing to the lack of a DNA extraction step. Therefore, these tests could be applied to small volume samples as an alternative to conventional gene doping tests.

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the \"metabolism\" and the \"metabolism of lipids\" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction.

Exercise plays an important role in regulating energy homeostasis, which affects the diversity of the intestinal microbial community in humans and animals. To the best of the authors\' knowledge, few studies have reported the associations between horse gut microbiota along with their predicted metabolic activities and the athletic ability of Jeju horses and Thoroughbreds living in Korea. This study was conducted to investigate the association between the gut microbiota and athletic performance in horses. This study sequenced the V3 and V4 hypervariable regions of the partial 16S rRNA genes obtained from racehorse fecal samples and compared the fecal microbiota between high- and low-performance Jeju horses and Thoroughbreds. Forty-nine fecal samples were divided into four groups: high-performance Jeju horses (HJ, n = 13), low-performance Jeju horses (LJ, n = 17), high-performance Thoroughbreds (HT, n = 9), and low-performance Thoroughbreds (LT, n = 10). The high-performance horse groups had a higher diversity of the bacterial community than the low-performance horse groups. Two common functional metabolic activities of the hindgut microbiota (i.e., tryptophan and succinate syntheses) were observed between the low-performance horse groups, indicating dysbiosis of gut microbiota and fatigue from exercise. On the other hand, high-performance horse groups showed enriched production of polyamines, butyrate, and vitamin K. The racing performance may be associated with the composition of the intestinal microbiota of Jeju horses and Thoroughbreds in Korea.

BACKGROUND: Racehorses commonly train and race in one direction, which may result in gait asymmetries. This study quantified gait symmetry in two cohorts of Thoroughbreds differing in their predominant exercising direction; we hypothesized that there would be significant differences in the direction of asymmetry between cohorts.
METHODS: 307 Thoroughbreds (156 from Singapore Turf Club (STC)-anticlockwise; 151 from Hong Kong Jockey Club (HKJC)-clockwise) were assessed during a straight-line, in-hand trot on firm ground with inertial sensors on their head and pelvis quantifying differences between the minima, maxima, upward movement amplitudes (MinDiff, MaxDiff, UpDiff), and hip hike (HHD). The presence of asymmetry (≥5 mm) was assessed for each variable. Chi-Squared tests identified differences in the number of horses with left/right-sided movement asymmetry between cohorts and mixed model analyses evaluated differences in the movement symmetry values.
RESULTS: HKJC had significantly more left forelimb asymmetrical horses (Head: MinDiff p < 0.0001, MaxDiff p < 0.03, UpDiff p < 0.01) than STC. Pelvis MinDiff (p = 0.010) and UpDiff (p = 0.021), and head MinDiff (p = 0.006) and UpDiff (p = 0.017) values were significantly different between cohorts; HKJC mean values indicated left fore- and hindlimb asymmetry, and STC mean values indicated right fore- and hindlimb asymmetry.
CONCLUSIONS: the asymmetry differences between cohorts suggest that horses may adapt their gait to their racing direction, with kinematics reflecting reduced \'outside\' fore- and hindlimb loading.

Jumps racing is a form of Thoroughbred horse racing that involves hurdles and steeples and typically longer distances, and heavier weights compared with flat racing, which does not incorporate obstacles. In Australia, jumps racing is carried out only in Victoria, one of eight states and territories. The continuation of jumps racing is contentious due to the higher risk of fatalities, falls and injuries for horses, compared with flat racing. While measures have been introduced by the industry to improve the safety of riders and horses, the rates of fatalities, falls and injuries in horses participating in jumps races have not been collectively reported in Australia since the 2012 to 2014 race seasons. Although information on individual horse fatalities, falls and injuries is published by Racing Victoria in Stewards\' Reports, the data are not aggregated, and so cannot readily be used to assess trends or evaluate the efficacy of safety measures introduced by the industry. The aim of this study was to determine the fatality, fall and injury rates for horses participating in hurdle and steeplechase races in Victoria in the 2022 and 2023 Thoroughbred horse jumps racing seasons compared with horses participating in flat races at the same race meets. Data on horse fatalities, falls and injuries were extracted from the published Racing Victoria race results and Stewards\' Reports for the jumps races (n = 150) and corresponding flat races (n = 157) held at the 38 jumps race meets in Victoria in 2022 and 2023. Overall, horse fatalities, falls and injuries occurred at higher rates in jumps races compared with flat races during the study period. The rate of horse fatalities in jumps races was 3.3 per 1000 starts, with no fatalities in flat races. The rate of horse falls in hurdle races was 24 per 1000 starts and 41.6 per 1000 starts in steeplechase races, comparable with rates previously reported in the 2012 to 2014 seasons. There were no falls in flat races. Horse injuries occurred at a rate of 68.9 per 1000 starts in jumps races compared with 18.8 per 1000 starts in flat races. In hurdle and steeplechase races, veterinary clearance being required following horse injury was 5.4 times (OR 5.4, 95% CI 2.8-10.2) and 7.2 times (OR 7.2, 95% CI 3.3-15.6) more likely, respectively, compared with flat races. The risk of trauma was 4 times more likely in hurdle and steeplechase races (OR 4.8, 95% CI 1.7-13.3 and OR 4.1, 95% CI 1.2-13.4, respectively) and the risk of lameness was increased by 2.5 times in hurdles (OR 2.5, 95% CI 1.2-5.2) and 5.1 times in steeplechase races (OR 5.1, 95% CI 2.3-11.5), compared with flat races. These findings support concerns about the welfare of horses involved in jumps racing and of the need for further safety measures to reduce these risks.

Determining whether endometrial microbial isolates are pathogens, contaminants, or even part of the \"normal\" microbiome is extremely complex, particularly given the absence of \"gold standard\" tests for endometritis. Population-level benchmarking and temporal monitoring can provide novel insights and a wider context to improve understanding. This study aimed to (i) estimate the prevalence of endometrial isolates from swabs of Thoroughbred broodmares in Newmarket, UK between 2014 and 2020; and (ii) evaluate the effects of year, mare age, and cytology findings on isolate prevalence. Generalised linear mixed models with a logit link, both null models and models using year of sampling, mare age, or cytology findings as predictors, were fitted to estimate isolate prevalence. Over the 7-year period, data were available from 18,996 endometrial-swab samples from 6050 mares on 290 premises. The overall isolate prevalence was 35.5% (95% confidence interval (CI) 33.0-37.9), and this varied significantly between years. The most prevalent isolates were β-hemolytic Streptococcus (17.9; 95% CI: 17-19) and E. coli (10.3%; 95% CI: 9.0-11.6). Isolate prevalence increased with mare age except for E. coli isolates, and with increasing category of cytology findings except for α-hemolytic Streptococcus isolates. The results provide novel estimates of isolate prevalence and highlight knowledge gaps around potential complexities in the interpretation of findings.