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Abstract Identifying patients who would benefit from extensive catheter ablation along with pulmonary vein isolation (PVI) among those with persistent atrial fibrillation (AF) has been a subject of controversy. The objective of this study was to apply uplift modeling, a machine learning method for analyzing individual causal effect, to identify such patients in the EARNEST-PVI trial, a randomized trial in patients with persistent AF. We developed 16 uplift models using different machine learning algorithms, and determined that the best performing model was adaptive boosting using Qini coefficients. The optimal uplift score threshold was 0.0124. Among patients with an uplift score ≥ 0.0124, those who underwent extensive catheter ablation (PVI-plus) showed a significantly lower recurrence rate of AF compared to those who received only PVI (PVI-alone) (HR 0.40; 95% CI 0.19–0.84; P-value = 0.015). In contrast, among patients with an uplift score

Abstract It remains unknown whether the recent trend of short dual antiplatelet therapy (DAPT) followed by P2Y12 inhibitor monotherapy can simply be applied to patients undergoing complex percutaneous coronary intervention (PCI). We performed a systematic review and meta-analysis to evaluate P2Y12 inhibitor monotherapy vs. conventional DAPT in patients undergoing complex PCI and non-complex PCI (PROSPERO: CRD42022335723). Primary endpoint was the 1-year Net Adverse Clinical Event (NACE). Among 5,323 screened studies, six randomized trials fulfilled the eligibility criteria. A total of 10,588 complex PCI patients (5,269 vs. 5,319 patients) and 25,618 non-complex PCI patients (12,820 vs 12,798 patients) were randomly assigned to P2Y12 inhibitor monotherapy vs. conventional DAPT. In complex PCI patients, P2Y12 inhibitor monotherapy was associated with a lower risk of NACE than conventional DAPT [Odds ratio (OR) 0.76, 95% confidence interval (CI) 0.63–0.91, P = 0.003], whereas in non-complex PCI patients, P2Y12 inhibitor monotherapy was associated with a trend toward lowering the risk of NACE (OR 0.86, 95% CI 0.72–1.02, P = 0.09). This meta-analysis across randomized trials demonstrated that a strategy of short DAPT followed by P2Y12 inhibitor monotherapy reduces the risk of 1-year NACE in patients undergoing complex PCI.

Abstract Background Under‐sensing (US) in implantable loop recorders (ILRs) interferes with the accurate diagnosis of arrhythmia, but there is little information available on the details of US of ILRs. The aim of this study was to clarify the frequency of US in patients with ILRs and to investigate the predictors of US in ILRs prior to implantation. Methods and Results We studied 46 consecutive patients implanted ILR. During the mean follow‐up period of 499 ± 363 days, 15 events of US were observed in five patients. There were no significant differences in patient characteristics between patients with and without US. In standard 12‐lead electrocardiogram (ECG), QRS complex amplitude in anterolateral chest leads (V2 to V5) were significantly lower in patients with than without US (V2: 0.88 [0.66, 1.22] mV vs. 1.67 [1.23, 2.29] mV, p = .010 V3: 1.25 [1.00, 1.26] mV vs. 1.90 [1.41, 2.29] mV, p = .013; V4: 1.14 [0.96, 1.38] mV vs. 1.93 [1.65, 2.64] mV, p = .023; V5: 0.57 [0.50, 0.75] mV vs. 1.60 [1.20, 1.98] mV, p = .011, respectively). ROC curve analysis showed that cut‐off values of 1.30 mV of QRS complex amplitude in V2, 1.26 mV of that in V3, and 0.75 mV of that in V5 had moderate accuracy for predicting US (V2: sensitivity 68%, specificity 100%, area under the curve [AUC] 0.86; V3: sensitivity 85%, specificity 80%, AUC 0.85; V5: sensitivity 98%, specificity 80%, AUC 0.85, respectively). Conclusions US was observed in 10.9% patients with an ILR. QRS complex amplitude in anterolateral chest leads (V2 to V5) on ECG might be useful for predicting US in patients with ILRs.

Abstract Aims Application of the latent class analysis to acute heart failure with preserved ejection fraction (HFpEF) showed that the heterogeneous acute HFpEF patients can be classified into four distinct phenotypes with different clinical outcomes. This model‐based clustering required a total of 32 variables to be included. However, this large number of variables will impair the clinical application of this classification algorithm. This study aimed to identify the minimal number of variables for the development of optimal subphenotyping model. Methods and results This study is a post hoc analysis of the PURSUIT‐HFpEF study (N = 1095), a prospective, multi‐referral centre, observational study of acute HFpEF [UMIN000021831]. We previously applied the latent class analysis to the PURSUIT‐HFpEF dataset and established the full 32‐variable model for subphenotyping. In this study, we used the Cohen's kappa statistic to investigate the minimal number of discriminatory variables needed to accurately classify the phenogroups in comparison with the full 32‐variable model. Cohen's kappa statistic of the top‐X number of discriminatory variables compared with the full 32‐variable derivation model showed that the models with ≥16 discriminatory variables showed kappa value of >0.8, suggesting that the minimal number of discriminatory variables for the optimal phenotyping model was 16. The 16‐variable model consists of C‐reactive protein, creatinine, gamma‐glutamyl transferase, brain natriuretic peptide, white blood cells, systolic blood pressure, fasting blood sugar, triglyceride, clinical scenario classification, infection‐triggered acute decompensated HF, estimated glomerular filtration rate, platelets, neutrophils, GWTG‐HF (Get With The Guidelines‐Heart Failure) risk score, chronic kidney disease, and CONUT (Controlling Nutritional Status) score. Characteristics and clinical outcomes of the four phenotypes subclassified by the minimal 16‐variable model were consistent with those by the full 32‐variable model. The four phenotypes were labelled based on their characteristics as ‘rhythm trouble’, ‘ventricular‐arterial uncoupling’, ‘low output and systemic congestion’, and ‘systemic failure’, respectively. Conclusions The phenotyping model with top 16 variables showed almost perfect agreement with the full 32‐variable model. The minimal model may enhance the future clinical application of this clustering algorithm.

Abstract Aims The effectiveness of angiotensin‐converting enzyme inhibitors (ACE‐I) and angiotensin II receptor blockers (ARB) has not been demonstrated in patients with heart failure with preserved ejection fraction (HFpEF). We recently reported significant interaction between the use of ACE‐I and/or ARB (ACE‐I/ARB) and frailty on prognosis in patients with HFpEF. In the present study, we examined the association between ACE‐I/ARB and prognosis in patients with HFpEF stratified by the presence or absence of frailty. Methods and results We examined the association between the use of ACE‐I/ARB and prognosis according to the presence [Clinical Frailty Scale (CFS) ≥ 5] or absence (CFS ≤ 4) of frailty in patients with HFpEF in a post hoc analysis of registry data. Primary endpoint was the composite of all‐cause mortality and heart failure admission. Secondary endpoints were all‐cause mortality and heart failure admission. Of 1059 patients, median age was 83 years and 45% were male. Kaplan–Meier analysis showed that the risk of composite endpoint (log‐rank P = 0.001) and all‐cause death (log‐rank P = 0.005) in patients with ACE‐I/ARB was lower in those with CFS ≥ 5, but similar between patients with and without ACE‐I/ARB in patients with CFS ≤ 4 (composite endpoint: log‐rank P = 0.830; all‐cause death: log‐rank P = 0.192). In a multivariable Cox proportional hazards model, use of ACE‐I/ARB was significantly associated with lower risk of the composite endpoint [hazard ratio (HR) = 0.52, 95% confidence interval (CI) = 0.33–0.83, P = 0.005] and heart failure admission (HR = 0.45, 95% CI = 0.25–0.83, P = 0.010) in patients with CFS ≥ 5, but not in patients with CFS ≤ 4 (composite endpoint: HR = 1.41, 95% CI = 0.99–2.02, P = 0.059; heart failure admission: HR = 1.43, 95% CI = 0.94–2.18, P = 0.091). The association between ACE‐I or ARB and prognosis did not significantly differ by CFS (CFS ≤ 4: log‐rank P = 0.562; CFS ≥ 5: log‐rank P = 0.100, for with ACE‐I vs. ARB, respectively). Adjusted HRs for CFS 1–4 were higher than 1.0 but were

Background: Individualized treatment approach based on pre-procedural precise risk balance assessment between bleeding and thrombosis would be desirable for patients with myocardial infarction (MI) undergoing emergent percutaneous coronary intervention (PCI) in this ultra-short dual antiplatelet therapy era. We aimed to develop and validate a quick thrombosis/bleeding risk-balance assessment tool. Methods: We developed and validated a novel thrombosis/bleeding risk-balance assessment tool using individual patient data from the prospective multicenter MI registry. Individual risks of thrombosis and bleeding within 7 days of the index PCI were estimated using a multinomial logistic regression model. The model was developed in the derivation cohort (4554 patients enrolled during 2003–2009) and validated in the validation cohort (2215 patients during 2010–2014). Results: A total of 6769 patients (66 ± 12 years, 5175 men) were eligible in this analysis. Predictive performance of the multinomial logistic regression models for bleeding and thrombosis assessed by calibration plots was good both in the derivation and validation cohorts. The net predicted probability (NPP) was defined as predicted probability of bleeding event (%) – predicted probability of thrombotic event (%). The NPP successfully stratified patients into those with a higher risk of bleeding than thrombosis and those with a higher risk of thrombosis than bleeding. This finding was consistent between the derivation and validation cohorts. Conclusions: We have established the risk balance assessment model for bleeding and thrombosis. Pre-procedural quick and precise assessment of the risk balance may help a decision making of procedural strategy and antithrombotic regimens in STEMI/non-STEMI patients undergoing PCI.

Abstract Aims Frailty is associated with prognosis of cardiovascular diseases. However, the significance of frailty in patients with heart failure with preserved ejection fraction (HFpEF) remains to be elucidated. The purpose of this study was to examine the prognostic significance of the Clinical Frailty Scale (CFS) in real‐world patients with HFpEF using data from a prospective multicentre observational study of patients with HFpEF (PURSUIT‐HFpEF study). Method and Results We classified 842 patients with HFpEF enrolled in the PURSUIT‐HFpEF study into two groups using CFS. The registry enrolled patients hospitalized with a diagnosis of decompensated heart failure. Median age was 82 [interquartile range: 77, 87], and 45% of the patients were male. Of 842 patients, 406 were classified as high CFS (CFS ≥ 4, 48%) and 436 as low CFS (CFS ≤ 3, 52%). The primary endpoint was the composite of all‐cause mortality and heart failure admission. Secondary endpoints were all‐cause mortality and heart failure admission. Patients with high CFS were older (85 vs. 79 years, P

Objectives The prognostic significance of an afterload-integrated diastolic index, the ratio of diastolic elastance (Ed) to arterial elastance (Ea) (Ed/Ea=[E/e′]/[0.9×systolic blood pressure]), is valid for 1 year after discharge in older patients with heart failure with preserved ejection fraction (HFpEF). We aimed to clarify the association with changes in Ed/Ea from enrolment to 1 year and prognosis thereafter in patients with HFpEF.Setting A prospective, multicentre observational registry of collaborating hospitals in Osaka, Japan.Participants We enrolled 659 patients with HFpEF hospitalised for acute decompensated heart failure (men/women: 296/363). Blood tests and transthoracic echocardiography were performed before discharge and at 1 year after.Primary outcome measures All-cause mortality and/or re-admission for heart failure were evaluated after discharge.Results High Ed/Ea assessed before discharge was a significant prognostic factor during the first, but not the second, year after discharge in all-cause mortality or all-cause mortality and/or re-admission for heart failure. When re-analysis was performed using the value of Ed/Ea at 1 year after discharge, high Ed/Ea was significant for the prognosis during the second year for both end points (p=0.012 and p=0.033, respectively). The poorest mortality during 1‒2 years after enrolment was observed in those who showed a worsening Ed/Ea during the first year associated with larger left ventricular mass index and reduced left ventricular ejection fraction. In all-cause mortality and/or re-admission for heart failure, the event rate during 1‒2 years was highest in those with persistently high Ed/Ea even after 1 year.Conclusions Time-sensitive prognostic performance of Ed/Ea, an afterload-integrated diastolic index, was observed in older patients with HFpEF.Trial registration number UMIN000021831.

Background Modification of arrhythmogenic substrates with extensive ablation comprising linear and/or complex fractional atrial electrogram ablation in addition to pulmonary vein isolation (PVI‐plus) can theoretically reduce the recurrence of atrial fibrillation. The DR‐FLASH score (score based on diabetes mellitus, renal dysfunction, persistent form of atrial fibrillation, left atrialdiameter >45 mm, age >65 years, female sex, and hypertension) is reportedly useful for identifying patients with arrhythmogenic substrates. We hypothesized that, in patients with persistent atrial fibrillation, the DR‐FLASH score can be used to classify patients into those who require PVI‐plus and those for whom a PVI‐only strategy is sufficient. Methods and Results This study is a post hoc subanalysis of the a multicenter, randomized controlled, noninferiority trial investigating efficacy and safety of pulmonary vein isolation alone for recurrence prevention compared with extensive ablation in patients with persistent atrial fibrillation (EARNEST‐PVI trial). This analysis focuses on the relationship between DR‐FLASH score and the efficacy of different ablation strategies. We divided the population into 2 groups based on a DR‐FLASH score of 3 points. A total of 469 patients were analyzed. Among those with a DR‐FLASH score >3 (N=279), the event rate of atrial arrhythmia recurrence was significantly lower in the PVI‐plus arm than in the PVI‐only arm (hazard ratio [HR], 0.45 [95% CI, 0.28–0.72]; P3 and DR‐FLASH score ≤3 (P value for interaction=0.020). Conclusions The DR‐FLASH score is a useful tool for deciding the catheter ablation strategy for patients with persistent atrial fibrillation. Registration URL: https://clinicaltrials.gov; Unique identifier: NCT03514693.

AimsAs part of efforts to identify candidates for patient education aimed at decreasing mortality from acute myocardial infarction, we investigated the prevalence of pre-infarction angina and its predictors among comorbidities in patients who were hospitalized with acute myocardial infarction (MI).MethodsWe conducted a prospective multicenter observational registry of MI patients from 1998 to 2014 (N = 12,093). The present study investigated the prevalence of pre-infarction angina and its predictors among comorbidities with a logistic regression model. Pre-infarction angina was defined as chest pain/oppression observed within 1 month before the onset of MI but which lasted 6 h before the onset of MI, while 15% did so ≤6 h before. Patients with hypertension, diabetes, dyslipidemia, or a family history of MI had a higher probability of pre-infarction angina than those without. Elderly patients and those with a history of cerebrovascular disease were less likely to experience pre-infarction angina.ConclusionsAlmost half of MI patients in our registry experienced pre-infarction angina before MI onset. Patients with hypertension, diabetes, dyslipidemia, or a family history of MI had a higher probability of experiencing pre-infarction angina than those without.

Background Stent thrombosis (ST) remains a significant medical issue. In particular, longer‐term mortality and clinical predictors after ST occurrence have yet to be elucidated. Methods and Results This was a multicenter, retrospective, observational study. A total of 187 definite ST cases from January 2008 to December 2017 were enrolled, and the long‐term clinical outcomes were investigated. The primary outcome measure was the cumulative mortality after ST occurrence. In addition, independent predictors of mortality were assessed. Among the stent types causing ST, bare‐metal stent, first‐generation drug‐eluting stent, second‐generation drug‐eluting stent, and third‐generation drug‐eluting stent comprised 31.0%, 19.3%, 36.9%, and 6.4% of cases, respectively. Median duration from stent implantation to ST was 680.5 (interquartile range, 33.8–2450.5) days. Cumulative mortality was 14.6%, 17.4%, 21.2%, 24.4%, and 33.8% at 1, 2, 3, 5 and 10 years, respectively. The cumulative mortality did not significantly differ by type of stent, and mortality of late ST was higher than that of early ST and very late ST; however, it did not reach statistical significance after the multivariate analysis. Independent predictors of mortality were hemodialysis (hazard ratio [HR], 7.80; 95% CI, 3.07–19.81; P

Background The previous large‐scale randomized controlled trial showed that routine thrombus aspiration (TA) during percutaneous coronary intervention (PCI) was associated with an increased risk of stroke. However, real‐world clinical evidence is still limited. Methods and Results We investigated the association between manual TA and stroke risk during primary PCI in the OACIS (Osaka Acute Coronary Insufficiency Study) database (N=12 093). The OACIS is a prospective, multicenter registry of myocardial infarction. The primary end point of the present study is stroke at 7 days. A total of 9147 patients who underwent primary PCI within 24 hours of hospitalization were finally analyzed (TA group, n=4448, versus non‐TA group, n=4699 patients). TA was independently associated with risk of stroke at 7 days (odds ratio [OR], 1.92 [95% CI, 1.19‒3.12]; P=0.008) in the simple logistic regression model, while the multilevel random effects logistic regression model with hospital treated as a random effect showed that manual TA was not associated with incremental risk of stroke at 7 days (OR, 0.91 [95% CI, 0.71‒1.16]; P=0.435). The 7‐day stroke risk of manual TA was significantly heterogeneous in different institutions (P for interaction=0.007). Conclusions Manual TA during primary PCI for patients with acute myocardial infarction was independently associated with the overall increased risk of periprocedural stroke. However, this result was substantially skewed because of institution specific risk variation, suggesting that the periprocedural stroke may be preventable by prudent PCI procedure or appropriate periprocedural management. Registration URL: https://upload.umin.ac.jp/cgi‐open‐bin/ctr_e/ctr_view.cgi?recptno=R000005464. Unique identifier: UMIN000004575.

The impact of changes in nutritional status during hospitalization on prognosis in patients with heart failure with preserved ejection fraction (HFpEF) remains unknown. We examined the association between changes in the Geriatric Nutritional Risk Index (GNRI) and prognosis during hospitalization in patients with HFpEF stratified by nutritional status on admission. Nutritional status did and did not worsen in 348 and 349 of 697 patients with high GNRI on admission, and in 142 and 143 of 285 patients with low GNRI on admission, respectively. Kaplan–Meier analysis revealed no difference in risk of the composite endpoint, all-cause death, or heart failure admission between patients with high GNRI on admission whose nutritional status did and did not worsen. In contrast, patients with low GNRI on admission whose nutritional status did not worsen had a significantly lower risk of the composite endpoint and all-cause death than those who did. Multivariable analysis revealed that worsening nutritional status was independently associated with a higher risk of the composite endpoint and all-cause mortality in patients with low GNRI on admission. Changes in nutritional status during hospitalization were thus associated with prognosis in patients with malnutrition on admission, but not in patients without malnutrition among those with HFpEF.

Background: Estimated plasma volume status (ePVS) is a well-validated prognostic indicator in heart failure. However, it remains unclear whether ePVS has prognostic significance in patients with acute myocardial infarction (AMI). Moreover, there is no available information on its additive effect with the Global Registry of Acute Coronary Events (GRACE) risk score in AMI patients. Methods: Data were obtained from the Osaka Acute Coronary Insufficiency Study (OACIS) registry database. Patients whose data were available for ePVS derived from Hakim’s formula and the GRACE risk score were studied. The primary endpoints were in-hospital and 5-year mortality. Results: Of 3930 patients, 206 and 200 patients died during hospitalization and 5 years after discharge, respectively. After adjustment, ePVS remained an independent predictor of in-hospital death (OR:1.02, 95% CI: 1.00–1.04, p = 0.036), and 5-year mortality(HR:1.03, 95% CI: 1.01–1.04, p

Background The female preponderance in heart failure with preserved ejection fraction (HFpEF) is a distinguishing feature of this disorder, but the association of sex with degree of diastolic dysfunction and clinical outcomes among individuals with HFpEF remains unclear. Methods and Results We conducted a prospective, multicenter, observational study of patients with HFpEF (PURSUIT‐HFpEF [Prospective Multicenter Observational Study of Patients with Heart Failure with Preserved Ejection Fraction]: UMIN000021831). Between 2016 and 2019, 871 patients were enrolled from 26 hospitals (follow‐up: 399±349 days). We investigated sex‐related differences in diastolic dysfunction and postdischarge clinical outcomes in patients with HFpEF. The echocardiographic end point was diastolic dysfunction according to American Society of Echocardiography/European Association of Cardiovascular Imaging criteria. The clinical end point was a composite of all‐cause death and heart failure readmission. Women accounted for 55.2% (481 patients) of the overall cohort. Compared with men, women were older and had lower prevalence rates of hypertension, coronary artery disease, and chronic kidney disease. Women had diastolic dysfunction more frequently than men (52.8% versus 32.0%, P

Objective During follow-up time, the value of prognostic factors may change, especially in the elderly patients, and the altered extent may affect the prognosis. We aimed to clarify the significance of the ratio of diastolic elastance (Ed) to arterial elastance (Ea), (Ed/Ea=(E/e’)/(0.9×systolic blood pressure)), an afterload-integrated diastolic index, in relation to follow-up periods and other laboratory factors, on the prognosis of elderly patients with heart failure with preserved ejection fraction (HFpEF).Methods We studied 552 HFpEF patients hospitalised for acute decompensated heart failure (men/women: 255/297). Blood testing and transthoracic echocardiography were performed before discharge. The primary endpoint was all-cause mortality.Results During a median follow-up of 508 days, 88 patients (men/women: 39/49) had all-cause mortality. During the first year after discharge, Ed/Ea (p=0.045) was an independent prognostic factor in association with albumin (p

Circulating microRNAs (miRNAs) are stable in bodily fluids and are potential biomarkers of various diseases and physiological states. Although several studies have been conducted on humans to detect drug doping by miRNAs, research on drugs and miRNAs in horses is limited. In this study, circulating miRNAs in horses after hydrocortisone administration were profiled and variations in miRNAs affected by hydrocortisone administration during endogenous hydrocortisone elevation were examined. The miRNAs were extracted from thoroughbred horse plasma before and after hydrocortisone administration and subjected to small RNA sequencing and reverse transcription quantitative PCR (RT-qPCR). RT-qPCR validation was performed for the 20 miRNAs that were most affected by hydrocortisone administration. The effects of elevated endogenous hydrocortisone levels due to exercise and adrenocorticotropic hormone administration were also confirmed. The validation results showed that approximately half of the miRNAs showed the same significant differences as those obtained using small RNA sequencing. Among the twenty miRNAs, two novel miRNAs and miR-133a were found to vary differently between exogenous hydrocortisone administration and endogenous hydrocortisone elevation. This study provides basic knowledge regarding the circulating miRNA profile of horses after hydrocortisone administration and identifies three miRNAs that could potentially be used as biomarkers to detect hydrocortisone administration.

Individual identification and paternity testing are important for avoiding inbreeding in the management of small populations of wild and domestic animals. In horse racing industries, they are extremely important for identifying and registering individuals and doping control to ensure fair competition. In this study, we constructed an individual identification panel for horses by using insertion and deletion (INDEL) markers. The panel included 39 INDEL markers selected from a whole-genome INDEL database. Genotyping of 89 Thoroughbreds showed polymorphisms with minor allele frequencies (MAFs) of 0.180-0.489 in all markers. The total probability of exclusion for paternity testing, power of discrimination, and probability of identity were 0.9994271269, >0.9999999999, and 0.9999999987, respectively. The panel was applied to 13 trios (sires, dams, and foals), and no contradictions were observed in genetic inheritance among the trios. When this panel was applied to the trios (52 trios) containing false fathers, an average of 7.3 markers excluded parentage relationships. In addition, genomic DNA extracted from the urine of six horses was partially genotyped for 39 markers, and 6-28 markers were successfully genotyped. The newly constructed panel has two advantages: a low marker mutation rate compared with short tandem repeats and a genotyping procedure that is as simple as short tandem repeat typing compared with single nucleotide variant typing. This panel can be applied for individual identification, paternity determination, and urine-sample identification in Thoroughbred horses., Competing Interests: The authors declare no competing interests concerning this study, including patents, products in development, or marketed products., (©2023 Catalyst Unit.)

To ensure fair competition and sports integrity, gene doping is prohibited in horseracing and equine sports. One gene doping method is by administering exogenous genes, called transgenes, to postnatal animals. Although several transgene detection methods have been developed for horses, many are unsuitable for multiplex detection. In this proof-of-concept study, we developed a highly sensitive and multiplex transgene detection method using multiple πCode with identification patterns printed on the surface. The following steps were employed: (1) multiplex polymerase chain reaction amplification of 12 targeted transgenes in a single tube, (2) detection using a mixture of 12 probes labeled with different πCodes, and (3) median fluorescence intensity measurement of fluorescent πCodes. Twelve transgenes cloned into plasmid vectors were targeted, and 1500 copies of each plasmid were spiked into 1.5 mL of horse plasma. Subsequently, a novel method using πCode succeeded in detecting all the transgenes using their DNA extracts. Additionally, we detected the erythropoietin ( EPO ) transgene in blood samples from a horse administered solely with the EPO transgene using this method. Therefore, the πCode detection method is considered suitable for multitarget gene detection in gene doping tests.

Glucocorticoid preparations have anti-inflammatory effects, and are commonly used in the equine clinical setting; however, such treatments can cause a number of side effects. Adrenal insufficiency is an adverse effect induced by the suppression of adrenal function following drug administration. This study aimed to investigate the influence of two glucocorticoid preparations, dexamethasone and hydrocortisone, on adrenocortical function in horses. The usual doses of dexamethasone and hydrocortisone preparations in equine practice were administered intramuscularly to six horses, and peripheral blood was collected at different time points. Concentrations of dexamethasone and hydrocortisone in the plasma, before and after drug administration, were measured using liquid chromatography-tandem mass spectrometry. Considering circadian rhythms in endogenous hydrocortisone levels, hormone concentrations, before and after drug administration, were compared at the same time of the day. Plasma dexamethasone concentrations were below the limit of quantification at 72 hr post-administration. Plasma hydrocortisone concentrations were significantly lower from 1 to 72 hr after administration. After hydrocortisone preparation administration, plasma hydrocortisone levels were significantly higher until 9 hr, and significantly lower at 24 and 48 hr. The suppression rate of endogenous hydrocortisone ranged over 2.2-5.3% with dexamethasone treatment and 17.5-45.7% with hydrocortisone treatment. The study clearly indicated the effects of glucocorticoids on adrenocortical function in horses and provided basic knowledge about the selection and prescription of glucocorticoid preparations and setting the withdrawal times in equine clinical setting.

Gene doping, which is prohibited in horseracing and equestrian sports, can be performed by introducing exogenous genes, known as transgenes, into the bodies of postnatal animals. To detect exogenous genes, a method utilizing quantitative polymerase chain reaction (qPCR) with a hydrolysis probe was developed to test whole blood and plasma samples, thereby protecting the fairness of competition and the rights of stakeholders in horseracing and equestrian sports. Therefore, we aimed to develop sample storage methods suitable for A and B samples in gene doping tests using blood. For sample A, sufficient qPCR detection was demonstrated after refrigeration for 1 to 2 weeks post collection. For sample B, the following procedures were confirmed to be suitable for storage: 1) centrifugation after sample receipt, 2) frozen storage, 3) natural thawing at room temperature, and 4) centrifugation without mixing blood cell components. Our results indicated that long-term cryopreservation yielded good plasma components from frozen blood samples even though it destroyed blood cells, indicating its applicability to the gene doping test using sample B, which can be stored for later use. Sample storage procedures are as important as detection methods in doping tests. Therefore, the series of procedures that we evaluated in this study will contribute to the efficient performance of gene doping tests through qPCR using blood samples., (©2023 The Japanese Society of Equine Science.)

Thoroughbreds are some of the most famous racehorses worldwide and are currently animals of high economic value. To understand genomic variability in Thoroughbreds, we identified genome-wide insertions and deletions (INDELs) and obtained their allele frequencies in this study. INDELs were obtained from whole-genome sequencing data of 101 Thoroughbred racehorses by mapping sequence reads to the horse reference genome. By integrating individual data, 1,453,349 and 113,047 INDELs were identified in the autosomal (1-31) and X chromosomes, respectively, while 18 INDELs were identified on the mitochondrial genome, totaling 1,566,414 INDELs. Of those, 779,457 loci (49.8%) were novel INDELs, while 786,957 loci (50.2%) were already registered in Ensembl. The sizes of diallelic INDELs ranged from -286 to +476, and the majority, 717,736 (52.14%) and 220,672 (16.03%), were 1-bp and 2-bp variants, respectively. Numerous INDELs were found to have lower frequencies of alternative (Alt) alleles. Many rare variants with low Alt allele frequencies (<0.5%) were also detected. In addition, 5955 loci were genotyped as having a minor allele frequency of 0.5 and being heterogeneous genotypes in all the horses. While short-read sequencing and its mapping to reference genome is a simple way of detecting variants, fake variants may be detected. Therefore, our data help to identify true variants in Thoroughbred horses. The INDEL database we constructed will provide useful information for genetic studies and industrial applications in Thoroughbred horses, including a gene-editing test for gene-doping control and a parentage test using INDELs for horse registration and identification.

We evaluated the utility of single nucleotide polymorphism (SNP) markers for parentage testing in Breton (BR) and Percheron (PR) horses in Japan using the proposed International Society for Animal Genetics (P-ISAG) 147 SNP panel and 414 autosomal SNPs. Genomic DNA was extracted from 98 horses of two breeds, BR (n = 47) and PR (n = 51), and sequenced using next-generation sequencing. The average minor allele frequencies for the P-ISAG panel for BR and PR were 0.306 and 0.301, respectively. The combined probabilities of exclusion (PEs) given two parents and one offspring: exclude a relationship (PE01) and given one parent and one offspring: exclude their relationship (PE02) were over 0.9999 for both breeds. Using the P-ISAG panel, no exclusion or doubtful cases were identified in 35 valid parent-offspring pairs, suggesting that the P-ISAG panel is helpful for parentage verification in both breeds. In contrast, as 0.18% of falsely accepted parentages were observed in the parentage discovery cases, additional markers such as the combination of the P-ISAG panel and 414 autosomal SNPs (561-SNP set) presented here should be used to identify valid parent-offspring pairs of horses with unknown parentage relationships., (© 2023 Japanese Society of Animal Science.)

The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.

Processed pseudogenes, also known as retrocopy genes, are copies of messenger RNAs that have been reverse transcribed into DNA and inserted into the genome. In this study, we identified 62 processed pseudogene candidates as intron-less genes from whole-genome sequencing (WGS) data of Thoroughbred horses using delly structural variation software. The 62 processed pseudogene candidates were confirmed by PCR amplification of intron-less products. A total of 11 processed pseudogenes were confirmed in the genome of all 23 analysed horses, whereas three processed pseudogenes with structures of ATP11B, DPH3 and RPL17 were detected in only one of 115 horses by PCR amplification of intron-less products. Currently, most of the gene doping tests proposed in human and horse sports are adapted PCR-based methods using hydrolysis probes to detect exon/exon junctions in transgenes because the operation is simple and economical. However, when the pseudogene is present in the host genome, the PCR-based methods may have a potential risk of detecting false positives. In this study, because processed pseudogenes that exist less frequently in the horse genome may affect PCR-based transgene detection in gene-doping tests, we propose and demonstrate that PCR amplification and sequencing using primers designed on transgene and promotors and/or polyadenylation signal for gene expression are useful for gene-doping detection as an additional confirmatory test to prevent false positives., (© 2022 Stichting International Foundation for Animal Genetics.)

Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected low-copy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene-doping detection., (© 2021 John Wiley & Sons, Ltd.)

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 10 4 (standard deviation: 4.34 × 10 3 ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control., (© 2021 John Wiley & Sons, Ltd.)

One method of gene doping in horseracing is administering of exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs for reference materials (RMs) and/or positive template controls (PTCs) are necessary for simultaneous analysis of multiple transgene targets. In this study, we designed and developed a novel RM for simultaneously detecting multiple targets via microfluidic quantitative PCR (MFQPCR). Twelve equine genes were selected as targets in this study. A sequence region including primers and probes for quantitative PCR was designed, and a 10 bp sequence was inserted to allow the RM to be distinguished from the original transgene sequences. The sequences of individual detection sites were then connected for 12 genes and cloned into a single plasmid vector. We performed fragment size analysis to distinguish between the PCR products of the original transgene sequence and those of the RM, enabling identification of RM contamination. PTCs diluted to 10,000, 1,000, 100, and 10 copies/µl with horse genomic DNA from RM were stably stored at 4°C for 1 year. As digital PCR enabled absolute quantification, the designed substances can serve as an RM. These findings indicate that the RM design and storage conditions were suitable for gene doping tests using MFQPCR., (©2021 The Japanese Society of Equine Science.)

Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time PCR against gene doping. Digital PCR amplification was significantly inhibited by high concentrations of proteinase K (more than 0.1 μg/μl), ethylenediaminetetraacetic acid (more than 5 nmol/μl), and heparin (more than 0.05 unit/μl) but not by ethanol or genomic DNA. In addition, phenol affected droplet formation in the digital PCR amplification process. Real-time PCR amplification was inhibited by high concentrations of phenol (more than 1% v/v), proteinase K (more than 0.001 μg/μl), ethylenediaminetetraacetic acid (more than 1 nmol/μl), heparin (more than 0.005 unit/μl), and genomic DNA (more than 51.9 ng/μl) but not by ethanol. Although both PCR systems were inhibited by nearly the same substances, digital PCR was more robust than real-time PCR against the inhibitors. We believe that our findings are important for the development of better methods for transgene detection and prevention of false negative results in gene doping testing., (© 2021 John Wiley & Sons, Ltd.)

The Thoroughbred breed was formed by crossing Oriental horse breeds and British native horses and is currently used in horseracing worldwide. In this study, we constructed a single-nucleotide variant (SNV) database using data from 101 Thoroughbred racehorses. Whole genome sequencing (WGS) revealed 11,570,312 and 602,756 SNVs in autosomal (1-31) and X chromosomes, respectively, yielding a total of 12,173,068 SNVs. About 6.9% of identified SNVs were rare variants observed only in one allele in 101 horses. The number of SNVs detected in individual horses ranged from 4.8 to 5.3 million. Individual horses had a maximum of 25,554 rare variants; several of these were functional variants, such as non-synonymous substitutions, start-gained, start-lost, stop-gained, and stop-lost variants. Therefore, these rare variants may affect differences in traits and phenotypes among individuals. When observing the distribution of rare variants among horses, one breeding stallion had a smaller number of rare variants compared to other horses, suggesting that the frequency of rare variants in the Japanese Thoroughbred population increases through breeding. In addition, our variant database may provide useful basic information for industrial applications, such as the detection of genetically modified racehorses in gene-doping control and pedigree-registration of racehorses using SNVs as markers., (© 2021. The Author(s).)

Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan probe/primer sets for each one. A panel of 24 assays was prepared and detected via a microfluidic quantitative PCR (MFQPCR) system using integrated fluidic circuits (IFCs). The limit of detection of the panel was 6.25 copy/μL. Amplification-specificity was validated using several concentrations of reference materials and animal genomic DNA, leading to specific detection. In addition, target-specific detection was successfully achieved in a horse administered 20 mg of the EPO transgene via MFQPCR. Therefore, MFQPCR may be considered a suitable method for multiple-target detection in gene-doping control. To our knowledge, this is the first application of microfluidic qPCR (MFQPCR) for gene-doping control in horseracing.

Gene doping is prohibited in horseracing and equestrian sports. In previous studies, we developed non-targeted transgene and genome editing detection methods based on whole genome resequencing (WGR) using genomic DNA extracted from whole blood. In this study, we aimed to develop a WGR method using DNA extracts from hair roots. Hair roots are a preferred substrate because their collection is less invasive than blood collection. Hair is also easier to store for long periods of time. Although almost all genomic DNA extracted from hair root samples stored for years at room temperature was degraded, the quality of genomic DNA from samples stored for years at refrigerated temperatures (4-8°C) was maintained. High-molecular-weight genomic DNA was isolated from hair roots using a magnetic silica beads method of extraction, enabling WGR from horsehair root extracts. Nucleotide sequencing results and numbers of single-nucleotide polymorphisms and insertions/deletions concurred with those previously reported for WGR of DNA extracted from whole blood. Therefore, we consider that storing hair samples at refrigerated temperatures prevents degradation of DNA, allowing the detection of gene doping in these samples based on WGR. It is likely this finding will also have a deterrent effect, as it is now possible to test horses with archived samples even if they or their parents are deceased. To our knowledge, this is the first report employing WGR on horsehair roots stored for a long term., (©2020 The Japanese Society of Equine Science.)

Thoroughbred racehorses are produced by mating small numbers of Arabian stallions and native British mares, and have been improved by selection of horseracing performance for about 300 years. While these improvements led to good performance as racehorses, they exposed horses to numerous medical disorders, aggravated by extensive exercise. Fractures are frequent medical disorders in Thoroughbred racehorses. In this study, fracture heritability was estimated using 3,927 Japanese Thoroughbred racehorses to elucidate the risk of racehorse fractures. The heritability estimates of all examined fractures were low (h 2  = 0.06), while those of fractures in carpal bone and carpus (carpal bone plus distal radius) were moderate (h 2  = 0.37, 0.24, respectively). Fracture occurrence age for carpal bone and distal radius was both 3.3 years old and was younger than that for other fractures. These results indicated that a larger proportion of the variation in the studied population was due to genetic factors for carpal fractures than for other fractures, while the fractures at other bones were largely affected by environmental factors, correlated with the athlete period (number year in racing). These findings contribute to develop a management plan for suppressing racehorse fractures and improving horseracing safety., (© 2019 Blackwell Verlag GmbH.)

Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin ( EPO ) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the EPO transgene was cloned into a plasmid for use as a model. We extracted the spiked EPO transgene from horse plasma and urine via magnetic beads, followed by ddPCR amplification for absolute quantification and transgene detection. The results indicated high recovery rates (at least ~60% and ~40% in plasma and urine, respectively), suggesting successful detection of the spiked transgene at concentrations of >130 and 200 copies/mL of plasma and urine, respectively. Additionally, successful detection was achieved following intramuscular injection of 20 mg of the EPO transgene. This represents the first study demonstrating a method for detecting the EPO transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry., Competing Interests: There are no competing interests including patents, products in development, or marketed products to declare in relationship to this work.

The integrity of thoroughbreds is maintained under strict regulation involving DNA parentage testing, which is robust in a population with high genetic variability. The genetic variability of the thoroughbred population is possibly fluctuating because of selective breeding that has focused on adaptations for racing performance. To monitor genetic variability within the population and the effectiveness of short tandem repeat (STR) parentage testing, we investigated allele frequencies and the exclusion probability (PE) of 16-17 loci of a parentage panel in the Japanese thoroughbred population over 15 years. Expected heterozygosities (He) of 14 loci indicated a decreasing trend, and the average He of the population decreased significantly. Low genetic variability was possibly induced by a decrease in population size and a selective breeding bias. Four loci showed both a significant increase in allele frequency and a significant decrease in He; it is assumed that those loci were affected by positive selection for racing performance. There was a significant decrease in the PE because of the changes in genetic variability; however, it has remained over 0.99995. The current STR panel is still effective for parentage control, but it will be necessary to continuously monitor genetic variability, which has decreased over 15 years., (© 2018 Japanese Society of Animal Science.)

A routine parentage test as part of a conservation program for Kiso horses identified a possible sex chromosome anomaly in a 7 months-old filly because of an aberrant result using LEX3, an X-linked marker. We then analyzed X-linked markers (LEX26, TKY38, and TKY270), Y-linked markers (Eca.YH12, Eca.YM2, Eca.YA16, and the sex-determining region Y gene), and an X/Y marker (Amelogenin gene). This analysis demonstrated that the filly had not inherited an X chromosome from her sire. A karyotyping analysis confirmed that the filly was 63,XO. As it was suspected that the horse would be sterile, we avoided using the horse as a broodmare; the information should also serve to prevent unnecessary conflict between owners transferring and receiving the horse.

Objective: Clinical research on gene therapy has advanced the field of veterinary medicine, and gene doping, which is the illegal use of gene therapy, has become a major concern in horseracing. Since the International Federation of Horseracing Authorities defined the administration of oligonucleotides and its analogues as a genetic therapy in 2017, the development of therapeutic nucleotide-detection techniques has become an urgent need. Most currently marketed and developed oligonucleotide therapeutics for humans consist of modified nucleotides to increase stability, and phosphorothioate (PS) modification is common., Results: We demonstrated the specific detection of phosphorothioated oligonucleotides (PSOs) using LC/MS/MS. PSOs produce the specific product ion (m/z 94.9362) derived from PS moiety. PS is not derived from endogenous substances in animal body, and the product ion is a suitable marker for the detection of PSOs. With our strategy, reproducible target analyses were achieved for identifying the specific substances, with a LOD of 0.1 ng/mL and a quantification rage of 0.1-200 ng/mL in deproteinated plasma. Non-target analyses could also detect the presence of PSOs selectively with 100 ng/mL in the same matrix. These results suggested that the detection of PSOs in horse blood is possible by targeting the product ion using LC/MS/MS.

Objective: Doping control is an important and indispensable aspect of fair horse racing; genetic doping has been recently included to this. In this study, we aimed to develop a detection method of gene doping. A plasmid cloned with human erythropoietin gene (p.hEPO, 250 μg/head) was intramuscularly injected into a microminipig. Subsequently, p.hEPO was extracted from 1 mL of plasma and detected by droplet digital polymerase chain reaction., Results: The results confirmed that the maximum amount of plasmid was detected at 15 min after administration and the majority of the plasmid was degraded in the bloodstream within 1-2 days after administration. In contrast, low amounts of p.hEPO were detected at 2-3 weeks after administration. These results suggest that the proposed method to detect gene doping can help obtain information for experiments using horses.

The distribution of Y chromosomal haplotypes in Japanese native horse populations was investigated to obtain genetic information on these populations. Here, 159 male/gelded horses from eight local populations were investigated, and three Y haplotypes (JHT-1, JHT-2, and JHT-3) were identified by analyzing five Y-linked loci. Five populations had only JHT-1, whereas two populations had only JHT-2. One population had JHT-1 and JHT-3. Based on the geographical distribution of these haplotypes and previously reported haplotypes for other Asian horses, JHT-1 is considered to be a major haplotype in ancestral native horses. The fixation of each haplotype suggests the influence of independent breeding and genetic drift in each population. These findings complement the results from previous genetic studies of Japanese native horses.

Body weight is an important trait to confirm growth and development in humans and animals. In Thoroughbred racehorses, it is measured in the postnatal, training, and racing periods to evaluate growth and training degrees. The body weight of mature Thoroughbred racehorses generally ranges from 400 to 600 kg, and this broad range is likely influenced by environmental and genetic factors. Therefore, a genome-wide association study (GWAS) using the Equine SNP70 BeadChip was performed to identify the genomic regions associated with body weight in Japanese Thoroughbred racehorses using 851 individuals. The average body weight of these horses was 473.9 kg (standard deviation: 28.0) at the age of 3, and GWAS identified statistically significant SNPs on chromosomes 3 (BIEC2&#95;808466, P=2.32E-14), 9 (BIEC2&#95;1105503, P=1.03E-7), 15 (BIEC2&#95;322669, P=9.50E-6), and 18 (BIEC2&#95;417274, P=1.44E-14), which were associated with body weight as a quantitative trait. The genomic regions on chromosomes 3, 9, 15, and 18 included ligand-dependent nuclear receptor compressor-like protein (LCORL), zinc finger and AT hook domain containing (ZFAT), tribbles pseudokinase 2 (TRIB2), and myostatin (MSTN), respectively, as candidate genes. LCORL and ZFAT are associated with withers height in horses, whereas MSTN affects muscle mass. Thus, the genomic regions identified in this study seem to affect the body weight of Thoroughbred racehorses. Although this information is useful for breeding and growth management of the horses, the production of genetically modified animals and gene doping (abuse/misuse of gene therapy) should be prohibited to maintain horse racing integrity.

Transcriptome analyses based on DNA microarray technology have been used to investigate gene expression profiles in horses. In this study, we aimed to identify exercise-induced changes in the expression profiles of genes in the peripheral blood of Thoroughbred horses using DNA microarray technology (15,429 genes on 43,603 probes). Blood samples from the jugular vein were collected from six horses before and 1 min, 4 hr, and 24 hr after all-out running on a treadmill. After the normalization of microarray data, a total of 26,830 probes were clustered into four groups and 11 subgroups showing similar expression changes based on k-mean clustering. The expression level of inflammation-related genes, including interleukin-1 receptor type II (IL-1R2), matrix metallopeptidase 8 (MMP8), protein S100-A8 (S100-A8), and serum amyloid A (SAA), increased at 4 hr after exercise, whereas that of c-Fos (FOS) increased at 1 min after exercise. These results indicated that the inflammatory response increased in the peripheral blood cells after exercise. Our study also revealed the presence of genes that may not be affected by all-out exercise. In conclusion, transcriptome analysis of peripheral blood cells could be used to monitor physiological changes induced by various external stress factors, including exercise, in Thoroughbred racehorses.

Ligand-dependent nuclear receptor compressor-like (LCORL) encodes a transcription factor, and its polymorphisms are associated with measures of skeletal frame size and adult height in several species. Recently, the single nucleotide polymorphism (SNP) BIEC2-808543 located upstream of LCORL was identified as a genetic diagnostic marker associated with withers height in Thoroughbreds. In this study, 322 Thoroughbreds-in-training were genotyped for BIEC2-808543 to evaluate the association between genotype and body composition traits, including body weight, withers height, the ratio of body weight to withers height, chest circumference, and cannon circumference. Of these, withers height and cannon circumference were significantly associated with LCORL genotypes throughout almost the entire training period in males and females. Animals with a C/T genotype had higher withers height (maximum differences of 1.8 cm and 2.1 cm in males and females, respectively) and cannon circumstance (maximum differences of 0.65 cm and 0.48 cm in males and females, respectively) compared with animals with a T/T genotype. These results suggested that the regulation of LCORL expression influences the skeletal frame size in Thoroughbreds and thus, indirectly affects the body weight. Although LCORL and BIEC2-808543 would be useful for selective breeding in Thoroughbreds, the production of genetically modified animals and gene doping based on genetic information should be prohibited in order to maintain racing integrity.