Laboratory of Racing Chemistry

Horse racing is a unique sporting domain in which competitive fairness and pedigree authenticity are linked institutionally and inseparably [...].

• Taishu horses are an endangered Japanese breed whose genetics are insufficiently profiled. • Runs-of-homozygosity analysis indicated persistent and multi-generational inbreeding in the Taishu horse population. • Kinship estimation, MDS and GRM analyses revealed that all individuals formed one group. • We established the inbreeding status of Taishu horses, and our study methods potentially allow the identification of suitable candidates for introduction to this population. Taishu horses are an endangered Japanese breed facing challenges due to limited genetic diversity and insufficient profiling. In this study, we aimed to elucidate population genetics for this breed with a particular view to measures of inbreeding and relatedness that will support enhancement of the breed’s genetic diversity. We applied SNP-array techniques to assess inbreeding (runs of homozygosity [ROH], and F IS and F ROH indices) and relatedness (kinship, multi-dimensional scaling [MDS], and genetic relationship matrix [GRM]) in nearly all extant Taishu horses as of 2021 (n=48; age range: 1-27 years). Short ROH accounted for the majority of detected segments (22.75% of total ROH), while medium to very long ROH were present in all horses, indicating persistent, multi-generational inbreeding. Mean F IS and F ROH were -0.053 ± 0.098 and 0.194 ± 0.076, respectively. F IS and F ROH were broadly constant, irrespective of year of birth, which indicates a degree of success for the breeding program. Kinship, MDS, and GRM analyses identified close genetic relationships involving one horse of unknown pedigree and confirmed that all 48 individuals formed a single genetic cluster. This study establishes the inbreeding status of Taishu horses and demonstrates the utility of relatedness analyses, which could be used to evaluate suitable candidates for re-introduction into this population, and is of general interest to those involved in conservation efforts for other endangered equine breeds.

This study aimed to generate information for parentage testing in horse breeds using microsatellites (STRs) and single-nucleotide polymorphisms (SNPs). Genotype data were obtained from 189 horse hair root samples, including 38 Thoroughbreds (TBs), 17 Jeju horses (JHs), 20 Quarter horses (QHs), 21 American Miniatures (AMs), and 93 Mongolian horses (MHs), using 15 STR markers and 71 SNP markers. Comparative analysis revealed that the mean expected heterozygosity ranged from 0.468 (AM) to 0.491 (JH) for SNPs and from 0.695 (TB) to 0.791 (MH) for STRs. The mean observed heterozygosity ranged from 0.415 (AM) to 0.487 (MH) for SNPs and from 0.706 (JH) to 0.776 (MH) for STRs. The mean polymorphic information content ranged from 0.349 (AM) to 0.364 (MH) for SNPs and from 0.635 (TB) to 0.761 (MH) for STRs. The inbreeding coefficient ranged from -0.009 (MH) to 0.113 (AM) for SNPs and from -0.058 (TB) to 0.043 (AM) for STRs. The cumulative exclusion probability (PE) for the 71-SNP panel exceeded 0.9999, indicating that SNP markers may be sufficient for parentage testing. In comparison, the STR markers yielded a combined PE of 0.9988 when one parent was known and 0.9999 when both parents were known. These findings highlight the potential of SNPs as alternatives to STRs for routine paternity verification in horses.

Rationale To uphold the integrity of horseracing and equestrian sports, it is critical for an equine doping control laboratory to develop a comprehensive screening method to cover a wide range of target substances at the required detection levels in equine urine. Methods The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid‐phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed‐phase column and analyzed using liquid chromatography/high‐resolution mass spectrometry (LC/HRMS) in both electrospray ionization positive and negative modes in two separate injections. The analytical data were obtained in full scan and product ion scan (PIS) modes in an 11 min LC run. Results The method can detect 1011 compounds (in both positive and negative ion modes). Over 95% of the target compounds have limits of detections (LODs) ≤10 ng/mL, and more than 50% of the LODs are ≤0.5 ng/mL. The lowest LOD can reach down to 0.01 ng/mL. The applicability of the method was demonstrated by the successful detection of prohibited substances in overseas and domestic equine urine samples. Conclusions We have successfully developed a regular screening method for equine urine samples that can detect more than 1000 compounds at sub‐ppb levels in both positive and negative ion modes with full scan and PIS using LC/HRMS. Furthermore, this method can theoretically be expanded to accommodate an unlimited number of prohibited substances in full‐scan mode.

This study aimed to evaluate the pharmacokinetics (PK) of tranexamic acid (TXA) in horses and estimate its irrelevant plasma and urine concentrations using the pharmacokinetic/pharmacodynamic (PK/PD) approach by applying the Pierre-Louis Toutain model. TXA was intravenously administered to eight thoroughbred mares, and plasma and urine TXA concentrations were quantified by liquid chromatography/tandem mass spectrometry. The quantified data were used to calculate the PK parameters of TXA in horses. The plasma elimination curves were best-fitted to a three-compartment model. Using the Toutain model approach, irrelevant plasma and urine TXA concentrations were estimated to be 0.0206 and 0.997 μg/mL, respectively. The typical values of clearance, steady-state volume of distribution, and steady-state urine-to-plasma ratio were 0.080 L/kg/h, 0.86 L/kg, and 49.0, respectively. The obtained irrelevant concentrations will be useful for establishing relevant regulatory screening limits for effective control of TXA use in horse racing and equestrian sports.

Vadadustat is a newly launched hypoxia-inducible factor stabilizer with anti-anemia and erythropoietic effects; however, its use in horses is expressly forbidden in both racing and equestrian competitions. Following our previous report on the pharmacokinetic study of vadadustat in horse plasma and urine, a long-term longitudinal analysis of vadadustat in horse hair after nasoesophageal administration (3 g/day for 3 days) to three thoroughbred mares is described in this study. Our main objective is to further extend the detection period of vadadustat for the purpose of doping control. Three bunches of mane hair from each horse were collected at 0 (pre), 1, 2, 3 and 6 month(s) post-administration. These hair samples were each cut into 2-cm segments and pulverized after decontamination of hair samples. The analyte in the powdered hair samples was extracted with liquid-liquid extraction followed by further purification by solid-phase extraction with strong anion exchange columns. The amount of vadadustat incorporated into the hair was quantified with a newly developed and validated method using liquid chromatography-high-resolution mass spectrometry. Our results show that vadadustat was confirmed in all post-administration hair samples, but its metabolites were not present. Thus, the detection window for vadadustat could be successfully extended up to 6 months post-administration. Interestingly, the 2-cm segmental analysis revealed that the tip of the drug band in the hair shifted along with the hair shafts in correspondence with the average hair growth rate (∼2.5 cm/month) but gradually diffused more widely from 2 cm at 1 month post-administration to up to 14 cm at 6 months post-administration. However, the loss in the total amount of vadadustat in hair over time was observed to most likely be due to the degradation of vadadustat. These findings will be useful for the control of abuse and/or misuse of vadadustat and the interpretation of positive doping cases.

Individual identification and paternity testing are important for avoiding inbreeding inthe management of small populations of wild and domestic animals. In horse racingindustries, they are extremely important for identifying and registering individuals anddoping control to ensure fair competition. In this study, we constructed an individualidentification panel for horses by using insertion and deletion (INDEL) markers. The panelincluded 39 INDEL markers selected from a whole-genome INDEL database. Genotyping of 89Thoroughbreds showed polymorphisms with minor allele frequencies (MAFs) of 0.180–0.489 inall markers. The total probability of exclusion for paternity testing, power ofdiscrimination, and probability of identity were 0.9994271269, >0.9999999999, and0.9999999987, respectively. The panel was applied to 13 trios (sires, dams, and foals),and no contradictions were observed in genetic inheritance among the trios. When thispanel was applied to the trios (52 trios) containing false fathers, an average of 7.3markers excluded parentage relationships. In addition, genomic DNA extracted from theurine of six horses was partially genotyped for 39 markers, and 6–28 markers weresuccessfully genotyped. The newly constructed panel has two advantages: a low markermutation rate compared with short tandem repeats and a genotyping procedure that is assimple as short tandem repeat typing compared with single nucleotide variant typing. Thispanel can be applied for individual identification, paternity determination, andurine-sample identification in Thoroughbred horses.