Intelligence-based anti-doping via an Intelligence and Drug Testing Management (IDTM) system.

The Intelligence and Drug Testing Management (IDTM), a system that can enhance drug testing analytics with related horse information and intelligence in a single platform, can help identify and mitigate potential doping and other threats.

Analytical advances in horseracing medication and doping control from 2018 to 2023.

The analytical approaches taken by laboratories to implement robust and efficient regulation of horseracing medication and doping control are complex and constantly evolving. Each laboratory's approach will be dictated by differences in regulatory, economic and scientific drivers specific to their local environment. However, in general, laboratories will all be undertaking developments and improvements to their screening strategies in order to meet new and emerging threats as well as provide improved service to their customers. In this paper, the published analytical advances in horseracing medication and doping control since the 22nd International Conference of Racing Analysts and Veterinarians will be reviewed. Due to the unprecedented impact of COVID-19 on the worldwide economy, the normal 2-year period of this review was extended to over 5 years. As such, there was considerable ground to cover, resulting in an increase in the number of relevant publications included from 107 to 307. Major trends in publications will be summarised and possible future directions highlighted. This will cover developments in the detection of 'small' and 'large' molecule drugs, sample preparation procedures and the use of alternative matrices, instrumental advances/applications, drug metabolism and pharmacokinetics, the detection and prevalence of 'endogenous' compounds and biomarker and OMICs approaches. Particular emphasis will be given to research into the potential threat of gene doping, which is a significant area of new and continued research for many laboratories. Furthermore, developments in analytical instrumentation relevant to equine medication and doping control will be discussed.

PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry.

The term 'gene doping' is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: EPO, FST, GH1, IGF1, and ILRN1. The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplification from genomic DNA observed in some assays, a restriction digest step was introduced in the PCR protocol prior to cycling to cut the amplifiable targets within the endogenous genes. We made the screening stage of the test simpler and faster by multiplexing PCR assays for four transgenes (EPO, FST, IGF1, and ILRN1), while the GH1 assay is performed in simplex. Both stages of the test reliably detect at least 20 copies of each transgene in a background of genomic DNA equivalent to what is extracted from two milliliters of equine blood. The test protocol was documented and tested with equine blood samples provided by an official doping control authority. The developed tests will form the basis for screening official horseracing samples in Australia.

Fractures in Thoroughbred racing and the potential for pre-race identification of horses at risk.

Risk rates for and predisposing factors to fractures occurring in Thoroughbred racing that have been published in peer reviewed journals are documented. The potential for currently available techniques to identify horses at increased risk for fracture is discussed on the bases of principles, practicalities, advantages, disadvantages and current data. All are reviewed in light of justifiable decision making and importance of fractures to horseracing's social license.

Pharmacokinetics and metabolism of lidocaine HCl 2% with epinephrine in horses following a palmar digital nerve block.

BACKGROUND: Lidocaine is a local anesthetic that is sometimes administered in combination with epinephrine. The addition of epinephrine increases the time lidocaine remains at the site of administration, thus prolonging the duration of effect. Due to their potential to prevent the visual detection of lameness, the administration of local anesthetics is strictly regulated in performance and racehorses. Recent reports of positive regulatory findings for lidocaine in racehorses suggests a better understanding of the behavior of this drug is warranted. The objective of the current study was to describe serum and urine concentrations and the pharmacokinetics of lidocaine and its primary metabolites following administration in combination with epinephrine, as a palmar digital nerve block in horses. Twelve horses received a single administration of 1 mL of 2% lidocaine HCl (20 mg/horse) with epinephrine 1:100,000, over the palmar digital nerve. Blood samples were collected up to 30 h and urine samples up to 48 h post administration. Lidocaine and metabolite concentrations were determined by liquid chromatography- mass spectrometry and pharmacokinetic (non-compartmental and compartmental) analysis was performed. RESULTS: Serum concentrations of lidocaine and 3-hydroxylidocaine were above the LOQ of the assay at 30 h post administration and monoethylglycinexylidide (MEGX) and glycinexylidide (GX) were below detectable levels by 24 and 48 h, respectively. In urine, lidocaine, MEGX and GX were all non-detectable by 48 h post administration while 3-hydroxylidocaine was above LOQ at 48 h post administration. The time of maximal concentration for lidocaine was 0.26 h (median) and the terminal half-life was 3.78 h (mean). The rate of absorption (Ka) was 1.92 1/h and the rate of elimination (Kel) was 2.21 1/h. CONCLUSIONS: Compared to previous reports, the terminal half-life and subsequent detection time observed following administration of lidocaine in combination with epinephrine is prolonged. This is likely due to a decrease in systemic uptake of lidocaine because of epinephrine induced vasoconstriction. Results of the current study suggest it is prudent to use an extended withdrawal time when administering local anesthetics in combination with epinephrine to performance horses.

A 'worthy disciple of Galen', 'ardent sportsman' and 'expert swordsman': Henry Kipping (1726-1785) apothecary and surgeon at Brighton, England.

Henry Kipping (1726-1785) was an apothecary and surgeon in Brighton, England. Here we present a series of contemporary references to Kipping from newspaper, book, archive and web-based resources. Some relate to his medical practice (resuscitating a 'drowned' elderly physician and a fisherman, bleeding a member of parliament who had fallen from his horse and praising a nostrum for the 'gravel and stone'). Social references include a duel with an army officer whose sword Kipping confiscated. Kipping appears to have been popular, connected with members of Brighton's high society and passionate about traditional past times, e.g. swordsmanship, horse riding and hunting on the Sussex downs. Indeed, Kipping's horse ran in the earliest known horse race in Brighton (1770). He was consulted by notable local residents including the Thrale family of Brighton and Lady Wilhelmina Shelley (the latter evidenced by Kipping partaking in her funeral procession in 1772). Kipping lived and practised at 28 West street, a road most famous for its (now lost) George Inn where King Charles II stayed just prior to his escape to Normandy. Kipping comes across as a colourful and eccentric clinician.

Short Insertion and Deletion Discoveries via Whole-Genome Sequencing of 101 Thoroughbred Racehorses.

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.

Sustainability and the Thoroughbred Breeding and Racing Industries: An Enhanced One Welfare Perspective.

As society debates the use of animals in sport, entertainment, and leisure, there is an increasing focus on the welfare, social, and ecological impacts of such activities on the animals, human participants, people close to them, and the physical environment. This article introduces the "Enhanced One Welfare Framework" to reveal significant costs and benefits associated with Thoroughbred breeding and racing globally. In addition, relative to calls to ban horseracing and similar activities as part of sustainability approaches that focus chiefly on animals, the "Enhanced One Welfare Framework" is better positioned politically to guide discussions that renegotiate the conditions under which horses are used for sport and the impact racing has on humans and the planet. In 2020, the International Federation of Horseracing Authorities issued its minimum horse welfare standards based on the Five Domains model, positioning lifelong horse welfare as "fundamentally important to the viability and sustainability of the industry". In this article, we critique the One Welfare framework's historic lack of focus on sport and enhance it by including sport, leisure, and entertainment and framing it within the Five Domains model. We offer a novel extension of the Five Domains model beyond animal welfare to consider human welfare and the physical environmental impacts of the sport, leisure, and entertainment industries and propose innovations that may help thoroughbred breeding and racing assure a sustainable future.

Is Injury an Occupational Hazard for Horseracing Staff?

Occupational health is a key priority for the horseracing industry, yet little research on occupational injuries exists. This study investigated the prevalence and the effect of injury in British horseracing staff during a 12-month period. An online retrospective survey was answered by 352 participants, identifying self-reported injury prevalence, injury management practices and attitudes towards workplace injury reporting. Chi Squared tests for independence were undertaken. A total of 310 (88.1%) staff reported injuries; risk factors for injury type included self-perceived job security, working hours, and perceived job control. Physical limitations, loss of confidence, workplace changes, and lifestyle implications were reported as consequences of injury. A total of 75.3% (n = 134) of staff were likely to seek time-off following fractures, but only 48.6% (n = 86) would take time-off for concussion. Attitudes towards injury management were influenced by financial circumstances, perceived staff shortages, previous injury experiences, and perceived employer expectations. The high self-reported injury prevalence could result in decreased workforce efficiency, poor physical health, and negative implications on retention and career longevity. The perception of invisible injuries, i.e., concussion, and subsequent management, should be of immediate concern to racing organizations. This paper identifies recommendations to enhance the safety and wellbeing of horseracing staff.

Body Composition and Bone Health Status of Jockeys: Current Findings, Assessment Methods and Classification Criteria.

Jockeys are unlike other weight-making athletes as the sport of horse racing requires strict weight management to meet the racing stipulations, protracted working hours and an extended racing season with limited downtime. Several studies have reported on the body composition and bone status of male and female professional and retired jockeys, yet the variety of assessment techniques, lack of standardised testing protocols and classification inconsistency make interpretation and comparison between studies problematic. This review aimed to appraise the existing body composition and bone health evidence in jockeys and evaluate the assessment methods and classification criteria used. Dual-energy X-ray absorptiometry (DXA) has been used most frequently in jockey research to assess body composition and bone status, while various generic skinfold equations have been used to predict body fat percentage. Evidence indicates flat jockeys are now taller and heavier than the data reported in earlier studies. Absolute fat mass has steadily increased in male jockeys in the last decade. The bone status of male jockeys remains a concern as constant low bone density (BMD) is evident in a large percentage of young and experienced professional jockeys. Due to limited studies and variations in assessment methods, further research is required to investigate bone turnover markers in male and female jockeys. A standardised testing protocol using internationally recognised assessment guidelines is critical for the accurate interpretation and evaluation of body composition and bone health measurements. Furthermore, establishing jockey-specific BMD and bone turnover reference ranges should be considered using existing and future data.

Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control.

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.

Simulated validation of intron-less transgene detection using DELLY for gene-doping control in horse sports.

Gene doping is prohibited in horseracing. In a previous study, we developed a method for non-targeted transgene detection using DELLY, which is based on split-read (SR) and paired-end (PE) algorithms to detect structural variants, on WGS data. In this study, we validated the detection sensitivity of DELLY using artificially generated sequence data of 12 target genes. With DELLY, at least one intron was detected as a deletion in eight targeted genes using the 150 bp PE read WGS data, whereas all targeted genes were detected by DELLY using the 100 bp PE read data. The detection sensitivity was higher in 100 bp PE reads than in 150 bp PE reads, despite a lower total sequence coverage, probably because of mismatch tolerance between the mapped reads and reference genome. In addition, it was observed that the average intron size detected by SR alone was 293 bp and that that detected by both SR and PE was 8924 bp. Thus, we showed that transgenes with various intron-exon structures could be detected using DELLY, suggesting its application in gene-doping control in horses.

The Impact of COVID-19 on Staff Working Practices in UK Horseracing.

Due to COVID-19, horseracing was required to cease all activity in March 2020; however, little is known about the pandemic's impact on staff working practices. This study investigated the impact of COVID-19 on staff working practices during the initial lockdown phases. An online survey about working conditions during lockdown was answered by 287 participants. Chi-squared tests for independence and binary logistic regression (BLR) analysis was undertaken. A total of 53.7% (n = 154) of staff were working during lockdown. Pandemic-specific workplace changes were reported as effective by 87.8% (n = 115) of staff. Flat grooms reported workplace changes as less effective (χ2 (52, n = 131) = 92.996, p < 0.001). A total of 67.2% (n = 193) of staff were positive about job security. Trainers and grooms were significantly less likely to report jobs as secure (χ2 (52, n = 287) = 75.653, p < 0.05). The findings suggest that most of the racing industry positively received changes made by their employers to tackle the pandemic, and for staff still working during lockdown, their health and safety was prioritised. Continued development of employee support structures to promote job security and workforce stability is advised, which will minimise the disruption of staff changes on the care and welfare of the horses.

A candidate-SNP retrospective cohort study for fracture risk in Japanese Thoroughbred racehorses.

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.

Results of racetrack examinations of Standardbred horses at race meetings in New South Wales.

This study analysed the race day veterinary reports from harness racing meetings controlled by the New South Wales Greyhound and Harness Racing Regulatory Authority between 1 September 2008 and 30 June 2009. The findings of all prerace and postrace examinations were analysed, and the frequency of observations was recorded. Chi-square testing was used to determine whether the incidence of abnormalities differed between age groups and tracks. A total of 542 meetings were conducted during the period of the study, with veterinary examinations conducted at 395 of these meetings. A total of 520 veterinary examinations (60 prerace and 460 postrace) were conducted. During the period of the study, 3685 horses started 31,393 times. Injuries occurring during the warm-up or start were the most common finding on prerace examination (11 reports) followed by lameness (6 reports). Lameness was the most common problem identified during postrace examinations, occurring at a rate of 2.10 cases per 1000 starts. Other problems identified at a rate of more than 1 per 1000 starts were poor postrace recovery/heat stress (2.04) and lacerations (1.75). Falls occurred at a rate of 1.72 cases per 1000 starts. Two-year-old horses were less likely to be lame than 3, 5 and 6-year-old horses. There was no difference in the rate of falls or lameness between tracks. This study provides a benchmark against which ongoing improvements in welfare can be assessed.

Effect of different protocols on the mitigation of exercise-induced pulmonary hemorrhage in horses when administered 24 hours before strenuous exercise.

BACKGROUND: Public pressure exists in the United States to eliminate race-day furosemide administration despite its efficacy in decreasing the severity of equine exercise pulmonary hemorrhage (EIPH). No effective alternative prophylaxis strategies have been identified. OBJECTIVE: To investigate alternative protocols to race-day furosemide that might mitigate EIPH. ANIMALS: Seven fit Thoroughbreds with recent EIPH. METHODS: Double-blinded placebo-controlled Latin square crossover using a treadmill followed by a blinded placebo-controlled crossover study at a racetrack. First, horses exercised supramaximally to fatigue 24 hours after initiating 5 EIPH prophylaxis protocols: 0.5 and 1.0 mg/kg furosemide IV 24 hours pre-exercise with and without controlled access to water, and 24 hour controlled access to water. Effects were compared to those measured after giving a placebo 24 hours pre-exercise, and 0.5 mg/kg furosemide IV 4 hours pre-exercise. Bronchoalveolar lavage (BAL) erythrocyte count was determined 45-60 minutes postexercise after endoscopy to assign an EIPH score. Data were analyzed using linear mixed effects models. The most promising protocol from the treadmill study was further evaluated in 6 horses using endoscopy and BAL after 1100 m simulated races. RESULTS: Intravenous furosemide (0.5 mg/kg) administered 24 hours pre-exercise combined with controlled access to water decreased the severity of EIPH on the treadmill and at the racetrack. CONCLUSION AND CLINICAL IMPORTANCE: Administering 0.5 mg/kg furosemide 24 hours pre-racing combined with controlling water intake may be a strategy to replace race-day furosemide administration for the management of EIPH. A larger study is indicated to further evaluate whether this protocol significantly mitigates EIPH severity.

Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection.

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.

Whip Rule Breaches in a Major Australian Racing Jurisdiction: Welfare and Regulatory Implications.

Whip use in horseracing is increasingly being questioned on ethical, animal welfare, social sustainability, and legal grounds. Despite this, there is weak evidence for whip use and its regulation by Stewards in Australia. To help address this, we characterised whip rule breaches recorded by Stewards using Stewards Reports and Race Diaries from 2013 and 2016 in New SouthWales (NSW) and the Australian Capital Territory (ACT). There were more recorded breaches at Metropolitan (M) than Country (C) or Provincial (P) locations, and by riders of horses that finished first, second, or third than by riders of horses that finished in other positions. The most commonly recorded breaches were forehand whip use on more than five occasions before the 100-metre (m) mark (44%), and whip use that raises the jockey's arm above shoulder height (24%). It is recommended that racing compliance data be analysed annually to inform the evidence-base for policy, education, and regulatory change, and ensure the welfare of racehorses and racing integrity.

Two complementary methods to control gonadotropin-releasing hormone vaccination (Improvac®) misuse in horseracing: Enzyme-linked immunosorbent assay test in plasma and steroidomics in urine.

Since the availability on the European market of the vaccine Improvac® dedicated to male pig immunological castration, the risk of misuse of this product in horses is now considered as a threat for the horseracing industry. Immunological castration is not allowed by the racing codes (immune system, Article 6). Indeed, this vaccination against the hypothalamic hormone luteinizing hormone-releasing hormone or gonadotropin-releasing hormone (GnRH) will prevent the release from the anterior pituitary of luteinizing hormone and follicle stimulating hormone, which are required for the development and activity of gonads in males (testes) and female (ovaries) and therefore all their subsequent physiological functions. This treatment will induce a strong hormonal variation resulting in a behaviour modification of the animals. In this work, four male standardbreds treated with Improvac® vaccine (two intramuscular injections within 4 weeks) were studied. Monitoring of the total scrotal width showed a decrease of the scrotum size (37%) and production of anti-GnRH antibodies was detected up to 200 days after the first injection. Anti-GnRH antibodies were detected in plasma after caprylic acid precipitation followed by an enzyme-linked immunosorbent assay (ELISA) as a rapid and efficient screening method applicable to routine analysis. These results were correlated to a switch of the sexual status from male group to gelding/female group obtained by a steroidomic approach with urine based on ten endogenous compounds. Copyright © 2017 John Wiley & Sons, Ltd.

Physiological and health characteristics of ex-jockeys.

OBJECTIVES: The long-term health implications resulting from extreme and chronic weight cycling and the associated energy restricted lifestyle of a professional jockey remain unknown. The aim of the present study was to describe and evaluate the physiological and health characteristics of retired jockeys. DESIGN: Cross-sectional. METHODS: Retired male jockeys (n=28; age 59±6yr; height 1.65±0.03m; body mass 73.7±10.9kg; BMI 26.9±3.8kgm(-2)) participated. Body composition (by dual energy X-ray absorptiometry; DXA), resting metabolic rate (RMR), glucose metabolism (by oral glucose tolerance test; OGTT), lipids and thyroid function were assessed. All data were interpreted in relation to established age specific reference ranges for the particular assessment undertaken. RESULTS: Forty three percent of the retired jockeys were classified as overweight, while 21% were obese. RMR was calculated as 1447±201kcalday(-1). Mean total cholesterol was 5.93±1.5mmolL(-1) for those not on cholesterol lowering medication and 5.09±1.1mmolL(-1) for those currently taking medication. No abnormalities in glucose metabolism and thyroid function were found. CONCLUSIONS: Chronic weight cycling during a career as a professional jockey did not induce any health consequences in the retired jockeys assessed in this study, however enhanced weight gain and dyslipidaemia were evident. Results suggest jockeys should be tracked longitudinally throughout their racing career and beyond to further establish long-term physical health implications associated with the current lifestyle of a professional jockey.