Identification of metabolomic changes in horse plasma after racing by liquid chromatography-high resolution mass spectrometry as a strategy for doping testing.

Recently, the illegal use of novel technologies, such as gene and cell therapies, has become a great concern for the horseracing industry. As a potential way to control this, metabolomics approaches that comprehensively analyze metabolites in biological samples have been gaining attention. However, it may be difficult to identify metabolic biomarkers for doping because physiological conditions generally differ between resting and exercise states in horses. To understand the metabolic differences in horse plasma between the resting state at training centres and the sample collection stage after racing for doping test (SAD), we took plasma samples from these two stages (n=30 for each stage) and compared the metabolites present in these samples by liquid chromatography-high resolution mass spectrometry. This analysis identified 5,010 peaks, of which 1,256 peaks (approximately 25%) were annotated using KEGG analysis. Principal component analysis showed that the resting state and SAD groups had entirely different metabolite compositions. In particular, the levels of inosine, xanthosine, uric acid, and allantoin, which are induced by extensive exercise, were significantly increased in the SAD group. In addition, many metabolites not affected by extensive exercise were also identified. These results will contribute to the discovery of biomarkers for detecting doping substances that cannot be detected by conventional methods.

Individual identification of racehorses from urine samples using a 26-plex single-nucleotide polymorphism assay.

To construct a system for identifying individual horses from urine samples that are submitted for postracing doping tests, we developed a genotyping assay based on 26-plex single-nucleotide polymorphisms (SNPs). DNA was isolated from urine using a commercially available DNA/RNA extraction kit, and SNP genotyping was achieved with a SNaPshot(™) technique. DNA profiles including 26 SNPs were acquired from urine samples and blood/hair samples. Within the studied Thoroughbred population, the 26-plex assay showed a probability of identity of 5.80 × 10(-11). Compared to the conventional short tandem repeat assay, the SNP assay used less DNA, and the rate of successful genotyping was improved to 97% using aliquots of horse urine as small as 140 µL. The urinary DNA could be successfully genotyped under proper storage concerning refrigeration or freeze-thawing. This SNP assay can be used for individual identification when suspicious results are obtained from horse doping tests.

Sequence variants at the myostatin gene locus influence the body composition of Thoroughbred horses.

Myostatin is a member of the transforming growth factor-ß family with a key role in inhibition of muscle growth by negative regulation of both myoblast proliferation and differentiation. Recently, a genomic region on ECA18, which includes the MSTN gene, was identified as a candidate region influencing racing performance in Thoroughbreds. In this study, four SNPs on ECA18, g.65809482T>C, g.65868604G>T, g.66493737C>T, and g.66539967A>G, were genotyped in 91 Thoroughbred horses-in-training to evaluate the association between genotype and body composition traits, including body weight, withers height, chest circumference, cannon circumference, and body weight/withers height. Of these, statistically differences in body weight and body weight/withers height were associated with specific genotypes in males. Specifically, body weight/withers height showed statistically significant differences depending on genotype at g.658604G>T, g.66493737C>T, and g.66539967A>G (P<0.01) in males during the training period. Animals with a genotype associated with suitability for short-distance racing, C/C at g.66493737C>T, had the highest value (3.17 ± 0.05 kg·cm(-1)) for body weight/withers height in March, while those with a genotype associated with suitability for long-distance racing, T/T, had the lowest (2.99 ± 0.03 kg·cm(-1)). In females, the trends in the association of body weight/withers height with genotypes were similar to those observed in males. As the SNPs are not believed to be linked to coding variants in MSTN, these results suggest that regulation of MSTN gene expression influences skeletal muscle mass and hence racing performance, particularly optimum race distance, in Thoroughbred horses.

Construction and validation of parentage testing for thoroughbred horses by 53 single nucleotide polymorphisms.

We characterized the SNP 53 JPN System for parentage verification during horse registry. The SNP 53 JPN System was constructed using 53 highly polymorphic single nucleotide polymorphisms (SNPs), which were amplified and genotyped with 2 multiplex assays. The SNP 53 JPN System showed good resolution for 95 unrelated thoroughbreds, and the exclusion probability (PE01) for each SNP ranged from 11.5 to 23.0%, resulting in a total PE01 value of 99.996%. These results indicate that the SNP 53 JPN System is useful for parentage testing of thoroughbreds. Of the 53 SNPs, 8 SNPs could be used to exclude a pseudo parent and sib combination found using the 2006 International Society for Animal Genetics (ISAG) horse comparison test, as efficiently as the parentage testing systems using short tandem repeats (STRs). Thus, we concluded that the SNP 53 JPN System could provide sufficient and reliable information for routine parentage testing of thoroughbred.

Molecular analysis using mitochondrial DNA and microsatellites to infer the formation process of Japanese native horse populations.

To assess the genetic diversity of Japanese native horse populations, we examined seven such populations using mitochondrial DNA (mtDNA) and microsatellite analyses. Four reference populations of Mongolian horses and European breeds were employed as other equids. In the mtDNA analysis, the control region (D-loop) of 411 bp was sequenced, and 12 haplotypes with 33 variable sites were identified in the Japanese native horses. The phylogenetic tree constructed by haplogrouping and using worldwide geographic references indicated that the haplotypes of the Japanese native horses were derived from six equid clusters. Compared with the foreign populations, the Japanese native populations showed lower within-population diversity and higher between-population differentiation. Microsatellite analysis, using 27 markers, found an average number of alleles per locus of 9.6 in 318 native and foreign horses. In most native populations, the within-population diversity was lower than that observed in foreign populations. The genetic distance matrix based on allelic frequency indicated that several native populations had notably high between-population differentiation. The molecular co-ancestry-based genetic distance matrix revealed that the European populations were differentiated from the Japanese and Mongolian populations, and no clear groups could be identified among the Japanese native horse populations. The genetic distance matrices had few correlations with the geographic distribution of the Japanese native populations. Based on the results of both mtDNA and microsatellite analyses, it could be speculated that each native population was formed by the founder populations derived from Mongolian horses. The genetic construction of each population appears to have been derived from independent breeding in each local area since the time of population fission, and this was accompanied by drastic genetic drift in recent times. This information will help to elucidate the ancestry of Japanese native horses.