Gray is a dominant coat color phenotype in horses caused by a 4.6 kb tandem triplication within intron 6 of syntaxin 17 (STX17). The copy number variation (CNV) of the duplicated segment influences the graying rate. The rare G2 allele (CNV = 2) is associated with a slower graying rate compared to the common G3 allele (CNV = 3) and is also relevant to melanoma risk. Current assays are limited because long and accurate PCR (LA-PCR) detects only the presence or absence of duplications, while droplet digital PCR (ddPCR) cannot reliably distinguish certain genotypes such as G3/g and G2/G2. We constructed a stepwise workflow combining (i) multiplex real-time PCR targeting the duplication junction for rapid gray/non-gray screening, (ii) ddPCR for copy number estimation, and (iii) LA-PCR for confirmatory genotyping of ambiguous copy-number classes. Using real-time PCR, we screened 4596 Japanese Thoroughbreds aged 2–7 years, of which 4374 were classified as non-gray and 222 as gray. Based on age and coat appearance, 23 Gy candidates were prioritized for slow-gray evaluation and analyzed by ddPCR; one was classified as G2/g, and 22 as G3/g or G2/G2. LA-PCR detected a g-derived band in all 22 cases, confirming that they were G3/g. Pedigree analysis suggested that the G2 allele was transmitted through the maternal line and that this lineage was distinct from the previously reported Japanese slow-gray family. This workflow enables practical molecular discrimination among non-gray, common gray, and slow-gray genotypes, supporting the surveillance of rare G2 alleles in the Japanese Thoroughbred population.
We examined the pharmacokinetics of intravenous pridinol in six thoroughbred horses. Each horse received a single 20 mg dose of pridinol mesylate via the jugular vein, and plasma and urine samples were collected over 72 h. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify pridinol concentrations in plasma and urine, allowing for the calculation of pharmacokinetic parameters. A three-compartment model best fit the plasma elimination data. Using the Toutain model, irrelevant plasma and urine concentrations were estimated to be 0.00284 and 0.000612 ng/mL, respectively. Key pharmacokinetic parameters were clearance rate, 1.27 L/h/kg; steady-state volume of distribution, 2.07 L/kg; and steady-state urine-to-plasma ratio, 0.211. These findings can help establish regulatory thresholds for pridinol in horse racing and equestrian sports.
The Miyako horse is a native Japanese horse breed. As with other native Japanese horses, the number of Miyako horses decreased due to mechanization and motorization, which reduced their roles, with just 14 in 1980. Although their population had increased to 55 horses by 2021, a further increase in their numbers is required to avoid extinction. Recently, their breeding has involved natural mating during group grazing; therefore, pedigree management has been difficult, and individual identification has been inconclusive. With the aim of formulating an effective breeding plan, this study used microsatellites to confirm parent-offspring relationships and evaluate the genetic diversity over time. First, the combination of microsatellite genotypes identified misunderstood parent-offspring relationships in 35.3% of the existing individuals, and a correct family tree was reconstructed. Next, the number of alleles and observed and expected values of heterozygosity were calculated separately for the populations during periods of 1998-2012 and 2013-2020. The values were 4.2, 0.705, and 0.653 and 3.9, 0.633, and 0.603, respectively, indicating that genetic diversity according to all indices decreased during period of 2013-2020. This was probably because of the bias of stallions in the 2013-2020 population. Errors in pedigree information in a small population such as Miyako horses could increase the risk of inbreeding, and confirmation of parent-offspring relationships using genotypes may be beneficial. Additionally, to maintain diversity in future breeding, it is important to avoid bias, particularly among stallions, and to ensure offspring of various individuals who are as distantly related to each other as possible.
V 1− x Ti x O 2 thin films have been prepared on Si 3 N 4 /SiO 2 /Si substrates by the two-step process of (1) growth of VO 2 /TiO 2 thin films by metal-organic decomposition with carbothermal reduction, and (2) the interdiffusion between V and Ti atoms at the interface between the VO 2 and TiO 2 thin films during the growth of the VO 2 thin film. The abrupt resistance change and the thermal hysteresis due to the metal-insulator transition were gradually suppressed with increasing the firing temperature, and they completely disappeared for the V 0.65 Ti 0.35 O 2 thin film prepared at 650 °C. A relatively high TCR of −4.2%/K was obtained from the film over a wide temperature range of 20 °C–90 °C. V 0.65 Ti 0.35 O 2 microbolometers with a size of 5 μ m × 15 μ m were fabricated by photolithography and Ar ion milling, and a Si 3 N 4 /SiO 2 membrane under the microbolometer was formed with an aqueous KOH solution. The DC sensitivity of the fabricated microbolometer on the membrane was estimated to be 9876 W −1 and 20 times higher than that of the microbolometer on the Si 3 N 4 /SiO 2 /Si substrate. The V 0.65 Ti 0.35 O 2 microbolometer on the membrane is available for a terahertz detector operating over the wide temperature range.
In drug metabolism studies in horses, non-targeted analysis by means of liquid chromatography coupled with high-resolution mass spectrometry with data-dependent acquisition (DDA) has recently become increasingly popular for rapid identification of potential biomarkers in post-administration biological samples. However, the most commonly encountered problem is the presence of highly abundant interfering components that co-elute with the target substances, especially if the concentrations of these substances are relatively low. In this study, we evaluated the possibility of expanding DDA coverage for the identification of drug metabolites by applying intelligently generated exclusion lists (ELs) consisting of a set of chemical backgrounds and endogenous substances. Daprodustat was used as a model compound because of its relatively lower administration dose (100 mg) compared to other hypoxia-inducible factor stabilizers and the high demand in the detection sensitivity of its metabolites at the anticipated lower concentrations. It was found that the entire DDA process could efficiently identify both major and minor metabolites (flagged beyond the pre-set DDA threshold) in a single run after applying the ELs to exclude 67.7–99.0
