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.

The present study indicates that hypermethylation may be involved in the pathogenesis of oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

SummaryMost skeletal tissues are thought to adapt to the mechanical environment they experience. While this has been demonstrated for muscle and bone, previous studies in the mature horse have failed to demonstrate adaptation in the superficial digital flexor tendon (SDFT), which suffers a high frequency of injury. This study tested the hypothesis that imposed exercise during growth would result in an increase in SDFT cross-sectional area (CSA). Fourteen Thoroughbred foals were divided into 2 sex-matched groups. A control group received 4 h pasture exercise and an exercise group had the same amount of pasture exercise with an additional short period of treadmill exercise daily from age 2-15 months. Activity at pasture was assessed objectively using a visual system. There was no significant difference in pasture activity between groups, although males were more active than females. The exercise programme resulted in a significantly larger tendon CSA in the exercise group at several, but not all, timepoints, which may be attributed to levels of variance. However, there was a significantly greater rate of increase in tendon CSA as a function of time in the exercised compared to the control group. This is the first evidence to suggest that tendon development can be modulated by exercise during growth in the horse, potentially increasing the ability of tendon to withstand the rigours of later athletic activity.

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.

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