An intronic copy number variation in Syntaxin 17 determines speed of greying and melanoma incidence in Grey horses.

The Greying with age phenotype in horses involves loss of hair pigmentation whereas skin pigmentation is not reduced, and a predisposition to melanoma. The causal mutation was initially reported as a duplication of a 4.6 kb intronic sequence in Syntaxin 17. The speed of greying varies considerably among Grey horses. Here we demonstrate the presence of two different Grey alleles, G2 carrying two tandem copies of the duplicated sequence and G3 carrying three. The latter is by far the most common allele, probably due to strong selection for the striking white phenotype. Our results reveal a remarkable dosage effect where the G3 allele is associated with fast greying and high incidence of melanoma whereas G2 is associated with slow greying and low incidence of melanoma. The copy number expansion transforms a weak enhancer to a strong melanocyte-specific enhancer that underlies hair greying (G2 and G3) and a drastically elevated risk of melanoma (G3 only). Our direct pedigree-based observation of the origin of a G2 allele from a G3 allele by copy number contraction demonstrates the dynamic evolution of this locus and provides the ultimate evidence for causality of the copy number variation of the 4.6 kb intronic sequence.

Evaluation of circulating miRNAs in mares approaching parturition.

Circulating microRNAs (miRNAs) are stable in body fluids and can serve as biomarkers for various diseases and physiological states. Although pregnancy-related miRNAs have been identified in various mammals, studies on parturition-related circulating miRNAs in mares are limited. Therefore, this study aimed to identify parturition-related miRNAs and examine their potential applications in the prediction of parturition date. miRNAs were extracted from the plasma of Thoroughbred mares 30 days (295-326 days pregnant) and 5 (323-352 days pregnant) - 0 (328-357 days pregnant) days before parturition, followed by small RNA sequencing (small RNA-seq) and reverse transcription quantitative PCR (RT-qPCR). Additionally, we measured plasma progestin concentrations in mares using an enzyme-linked immunosorbent assay. Small RNA-seq data indicated that 18 miRNAs were affected by parturition proximity. Among the 18 miRNAs, two novel miRNAs and three known miRNAs (miR-361-3p, miR-483, and miR-99a) showed significant changes at 5-0 days before parturition compared with that at 30 days to parturition. Plasma progestin concentrations were higher at 5-3 days to parturition than at 30 days to parturition, and then decreased on the day of parturition. Conclusively, this study provides basic knowledge of parturition-related circulating miRNAs in mares, and identifies miRNAs that could potentially be used as biomarkers to predict parturition in mares.

A method for detecting gene doping in horse sports without DNA extraction.

Gene doping is prohibited in horse sports and can involve the administration of exogenous genes, called transgenes, to postnatal animals. Quantitative polymerase chain reaction (qPCR) methods have been developed to detect gene doping; however, these generally require DNA extraction from the plasma prior to qPCR. In this study, we developed two methods, direct droplet digital PCR (ddPCR) and nested ddPCR, to detect the equine erythropoietin (EPO) transgene without DNA extraction. Direct ddPCR used pretreated plasma and PCR to detect the EPO transgene spiked at 10 copies/µL. Nested ddPCR utilised pre-amplification using nontreated plasma, purification of PCR products and PCR to detect the EPO transgene spiked at 1 copy/µL in plasma. These methods successfully detected the EPO transgene after intramuscular injection into horses. Since each method has different detection sensitivity, the combined use of direct ddPCR for screening and nested ddPCR for confirmation may complement each other and prevent the occurrence of false positives, allowing the reliable detection of gene-doped substances. One advantage of these methods is the small amount of sample required, approximately 2.2-5.0 µl, owing to the lack of a DNA extraction step. Therefore, these tests could be applied to small volume samples as an alternative to conventional gene doping tests.

Non-Synonymous Substitutions in Cadherin 13, Solute Carrier Family 6 Member 4, and Monoamine Oxidase A Genes are Associated with Personality Traits in Thoroughbred Horses.

Retraining retired racehorses for various purposes can help correct behavioral issues. However, ensuring efficiency and preventing accidents present global challenges. Based on the hypothesis that a simple personality assessment could help address these challenges, the present study aimed to identify genetic markers associated with personality. Eight genes were selected from 18 personality-related candidate genes that are orthologs of human personality genes, and their association with personality was verified based on actual behavior. A total of 169 Thoroughbred horses were assessed for their tractability (questionnaire concerning tractability in 14 types of situations and 3 types of impressions) during the training process. Personality factors were extracted from the data using principal component analysis and analyzed for their association with single nucleotide variants as non-synonymous substitutions in the target genes. Three genes, CDH13, SLC6A4, and MAOA, demonstrated significant associations based on simple linear regression, marking the identification of these genes for the first time as contributors to temperament in Thoroughbred horses. All these genes, as well as the previously identified HTR1A, are involved in the serotonin neurotransmitter system, suggesting that the tractability of horses may be correlated with their social personality. Assessing the genotypes of these genes before retraining is expected to prevent problems in the development of a racehorse's second career and shorten the training period through individual customization of training methods, thereby improving racehorse welfare.

Changes in population structure and genetic diversity of Misaki horses between 2015 and 2020.

For the preservation of Misaki horses, changes in the population structure and genetic diversity of the horses for 5 years were analyzed using population and genotype data from 2015-2020. The microsatellite genotyping was performed, and the average number of alleles (Na), expected heterozygosity (He), and observed value (Ho) were calculated. Moreover, the average generation length (GL) was estimated from the population management record. Then, no significant differences in Na, He, and Ho were found between 2015 and 2020, suggesting their genetic diversity had been maintained for 5 years. Moreover, the average GL was estimated as 4.6 years. Compared to other native horses, a short average GL suggesting a rapid generation renewing is a characteristic of the Misaki population.

Genome-wide search reveals the uniqueness of DNA regions associated with coat color and innate immunity in Hokkaido Native Horse.

Hokkaido Native Horse (HKD) is a horse breed native to Hokkaido in Japan known for the traits such as coat color with no white spots and adaptability to the local cold climate. To examine whether those traits of HKD are conferred at the DNA level, we attempted to identify fixed DNA regions in HKD individuals, that is, the selection signatures of HKD. A comparison of genome-wide single nucleotide polymorphism genotypes in 58 HKD individuals by principal component analysis, and cluster analysis between breeds, including HKD, and within the HKD individuals indicated the genetic independence of HKD as a breed. Tajima's D analysis and runs of homozygosity analysis identified 23 selection signatures unique to HKD (P < 0.05), and following database search found 20 traits that were associated with those selection signatures; among these traits, coat color traits, face and body markings, showed the highest important value (0.50 and 0.46). Enrichment analysis of genes in the selection signatures identified six gene ontology terms (P < 0.05), and a term related to innate immunity (regulation of defense response; GO:0031347) showed the highest positive fold enrichment value (7.13). These results provide the first scientific evidence of a genetic basis for the traits of HKD.

Construction of an individual identification panel for horses using insertion and deletion markers.

Individual identification and paternity testing are important for avoiding inbreeding in the management of small populations of wild and domestic animals. In horse racing industries, they are extremely important for identifying and registering individuals and doping control to ensure fair competition. In this study, we constructed an individual identification panel for horses by using insertion and deletion (INDEL) markers. The panel included 39 INDEL markers selected from a whole-genome INDEL database. Genotyping of 89 Thoroughbreds showed polymorphisms with minor allele frequencies (MAFs) of 0.180-0.489 in all markers. The total probability of exclusion for paternity testing, power of discrimination, and probability of identity were 0.9994271269, >0.9999999999, and 0.9999999987, respectively. The panel was applied to 13 trios (sires, dams, and foals), and no contradictions were observed in genetic inheritance among the trios. When this panel was applied to the trios (52 trios) containing false fathers, an average of 7.3 markers excluded parentage relationships. In addition, genomic DNA extracted from the urine of six horses was partially genotyped for 39 markers, and 6-28 markers were successfully genotyped. The newly constructed panel has two advantages: a low marker mutation rate compared with short tandem repeats and a genotyping procedure that is as simple as short tandem repeat typing compared with single nucleotide variant typing. This panel can be applied for individual identification, paternity determination, and urine-sample identification in Thoroughbred horses.

Identification of Potential miRNA Biomarkers to Detect Hydrocortisone Administration in Horses.

Circulating microRNAs (miRNAs) are stable in bodily fluids and are potential biomarkers of various diseases and physiological states. Although several studies have been conducted on humans to detect drug doping by miRNAs, research on drugs and miRNAs in horses is limited. In this study, circulating miRNAs in horses after hydrocortisone administration were profiled and variations in miRNAs affected by hydrocortisone administration during endogenous hydrocortisone elevation were examined. The miRNAs were extracted from thoroughbred horse plasma before and after hydrocortisone administration and subjected to small RNA sequencing and reverse transcription quantitative PCR (RT-qPCR). RT-qPCR validation was performed for the 20 miRNAs that were most affected by hydrocortisone administration. The effects of elevated endogenous hydrocortisone levels due to exercise and adrenocorticotropic hormone administration were also confirmed. The validation results showed that approximately half of the miRNAs showed the same significant differences as those obtained using small RNA sequencing. Among the twenty miRNAs, two novel miRNAs and miR-133a were found to vary differently between exogenous hydrocortisone administration and endogenous hydrocortisone elevation. This study provides basic knowledge regarding the circulating miRNA profile of horses after hydrocortisone administration and identifies three miRNAs that could potentially be used as biomarkers to detect hydrocortisone administration.

Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests.

Gene doping, which is prohibited in horseracing and equestrian sports, can be performed by introducing exogenous genes, known as transgenes, into the bodies of postnatal animals. To detect exogenous genes, a method utilizing quantitative polymerase chain reaction (qPCR) with a hydrolysis probe was developed to test whole blood and plasma samples, thereby protecting the fairness of competition and the rights of stakeholders in horseracing and equestrian sports. Therefore, we aimed to develop sample storage methods suitable for A and B samples in gene doping tests using blood. For sample A, sufficient qPCR detection was demonstrated after refrigeration for 1 to 2 weeks post collection. For sample B, the following procedures were confirmed to be suitable for storage: 1) centrifugation after sample receipt, 2) frozen storage, 3) natural thawing at room temperature, and 4) centrifugation without mixing blood cell components. Our results indicated that long-term cryopreservation yielded good plasma components from frozen blood samples even though it destroyed blood cells, indicating its applicability to the gene doping test using sample B, which can be stored for later use. Sample storage procedures are as important as detection methods in doping tests. Therefore, the series of procedures that we evaluated in this study will contribute to the efficient performance of gene doping tests through qPCR using blood samples.

Evaluation of parentage testing using single nucleotide polymorphism markers for draft horses in Japan.

We evaluated the utility of single nucleotide polymorphism (SNP) markers for parentage testing in Breton (BR) and Percheron (PR) horses in Japan using the proposed International Society for Animal Genetics (P-ISAG) 147 SNP panel and 414 autosomal SNPs. Genomic DNA was extracted from 98 horses of two breeds, BR (n = 47) and PR (n = 51), and sequenced using next-generation sequencing. The average minor allele frequencies for the P-ISAG panel for BR and PR were 0.306 and 0.301, respectively. The combined probabilities of exclusion (PEs) given two parents and one offspring: exclude a relationship (PE01) and given one parent and one offspring: exclude their relationship (PE02) were over 0.9999 for both breeds. Using the P-ISAG panel, no exclusion or doubtful cases were identified in 35 valid parent-offspring pairs, suggesting that the P-ISAG panel is helpful for parentage verification in both breeds. In contrast, as 0.18% of falsely accepted parentages were observed in the parentage discovery cases, additional markers such as the combination of the P-ISAG panel and 414 autosomal SNPs (561-SNP set) presented here should be used to identify valid parent-offspring pairs of horses with unknown parentage relationships.

Multiplex Detection of Transgenes Using πCode Technology for Gene Doping Control.

To ensure fair competition and sports integrity, gene doping is prohibited in horseracing and equine sports. One gene doping method is by administering exogenous genes, called transgenes, to postnatal animals. Although several transgene detection methods have been developed for horses, many are unsuitable for multiplex detection. In this proof-of-concept study, we developed a highly sensitive and multiplex transgene detection method using multiple πCode with identification patterns printed on the surface. The following steps were employed: (1) multiplex polymerase chain reaction amplification of 12 targeted transgenes in a single tube, (2) detection using a mixture of 12 probes labeled with different πCodes, and (3) median fluorescence intensity measurement of fluorescent πCodes. Twelve transgenes cloned into plasmid vectors were targeted, and 1500 copies of each plasmid were spiked into 1.5 mL of horse plasma. Subsequently, a novel method using πCode succeeded in detecting all the transgenes using their DNA extracts. Additionally, we detected the erythropoietin (EPO) transgene in blood samples from a horse administered solely with the EPO transgene using this method. Therefore, the πCode detection method is considered suitable for multitarget gene detection in gene doping tests.

Evaluation of genetic diversity using 31 microsatellites in Miyako horses.

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.

Evaluation of the effect of glucocorticoid treatment on adrenocortical functions by monitoring endogenous hydrocortisone in horses.

Glucocorticoid preparations have anti-inflammatory effects, and are commonly used in the equine clinical setting; however, such treatments can cause a number of side effects. Adrenal insufficiency is an adverse effect induced by the suppression of adrenal function following drug administration. This study aimed to investigate the influence of two glucocorticoid preparations, dexamethasone and hydrocortisone, on adrenocortical function in horses. The usual doses of dexamethasone and hydrocortisone preparations in equine practice were administered intramuscularly to six horses, and peripheral blood was collected at different time points. Concentrations of dexamethasone and hydrocortisone in the plasma, before and after drug administration, were measured using liquid chromatography-tandem mass spectrometry. Considering circadian rhythms in endogenous hydrocortisone levels, hormone concentrations, before and after drug administration, were compared at the same time of the day. Plasma dexamethasone concentrations were below the limit of quantification at 72 hr post-administration. Plasma hydrocortisone concentrations were significantly lower from 1 to 72 hr after administration. After hydrocortisone preparation administration, plasma hydrocortisone levels were significantly higher until 9 hr, and significantly lower at 24 and 48 hr. The suppression rate of endogenous hydrocortisone ranged over 2.2-5.3% with dexamethasone treatment and 17.5-45.7% with hydrocortisone treatment. The study clearly indicated the effects of glucocorticoids on adrenocortical function in horses and provided basic knowledge about the selection and prescription of glucocorticoid preparations and setting the withdrawal times in equine clinical setting.

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.

Identification of Personality-Related Candidate Genes in Thoroughbred Racehorses Using a Bioinformatics-Based Approach Involving Functionally Annotated Human Genes.

Considering the personality traits of racehorses (e.g., flightiness, anxiety, and affability) is considered essential to improve training efficiency and decrease accident frequency, especially when retraining for a second career that may involve contact with inexperienced personnel after retiring from racing. Studies on human personality-related genes are frequently conducted; however, such studies are rare in horses because a consistent methodology for personality evaluation is lacking. Using the recently published whole genome variant database of 101 Thoroughbred horses, we compared horse genes orthologous to human genes related to the Big Five personality traits, and identified 18 personality-related candidate genes in horses. These genes include 55 variants that involve non-synonymous substitutions that highly impact the encoded protein. Moreover, we evaluated the allele frequencies and functional impact on the proteins in terms of the difference in molecular weights and hydrophobicity levels between reference and altered amino acids. We identified 15 newly discovered genes that may affect equine personality, but their associations with personality are still unclear. Although more studies are required to compare genetic and behavioral information to validate this approach, it may be useful under limited conditions for personality evaluation.

Genetic characterization of Japanese native horse breeds by genotyping variants that are associated with phenotypic traits.

Concerns have been raised about the loss of genetic diversity in Japanese native horses because of their declining populations. In this study, we investigated the genetic variation of four genes, myostatin (MSTN), ligand-dependent nuclear receptor corepressor like (LCORL), doublesex and mab-3 related transcription factor 3 (DMRT3), and 5-hydroxytryptamine receptor 1A (HTR1A), which are associated with horse phenotypic traits, in six Japanese horse breeds (Hokkaido, Kiso, Noma, Misaki, Tokara, and Yonaguni). MSTN, LCORL, DMRT3, and HTR1A showed polymorphisms in the Kiso; Hokkaido and Noma; Hokkaido; and Kiso, Tokara, and Yonaguni breeds, respectively. The Misaki did not show polymorphisms in any of the genes. This study may serve as a basis for developing future breeding strategies focusing on traits in Japanese native horses.

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control.

The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.

Use of mitochondrial sequencing to detect gene doping in horses via gene editing and somatic cell nuclear transfer.

Gene editing and subsequent cloning techniques offer great potential not only in genetic disease correction in domestic animals but also in livestock production by enhancement of desirable traits. The existence of the technology, however, leaves it open to potential misuse in performance-led sports such as horseracing and other equestrian events. Recent advances in equine gene editing, regarding the generation of gene-edited embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer, have highlighted the need to develop tools to detect potential prohibited use of the technology. One possible method involves the characterisation of the mitochondrial genome (which is not routinely preserved during cloning) and comparing it with the sequence of the registered dam. We present here our approach to whole-mitochondrial sequencing using tiled long-range PCR and next-generation sequencing. To determine whether the background mutation rate in the mitochondrial genome could potentially confound results, we sequenced 10 sets of dam and foal duos. We found variation between duos but none within duos, indicating that this method is feasible for future screening systems. Analysis of WGS data from over 100 Thoroughbred horses revealed wide variation in the mitochondria sequence within the breed, further displaying the utility of this approach.

A comprehensive and comparative proteomic analysis of horse serum proteins in colitis.

BACKGROUND: Equine colitis is a diarrhoeal disease caused by inflammation of the large bowel and can potentially be life-threatening due to its rapid progression. Pathogenesis is multifactorial and pathophysiology is highly complicated, therefore, reliable diagnostic biomarkers are needed in the veterinary field. OBJECTIVE: Serum is one of the most commonly used diagnostic tools in equine clinical investigation. To discover diagnostic or prognostic protein markers for colitis in horse serum, comprehensive and comparative proteomic analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). STUDY DESIGN: Case-control study. METHODS: Serum samples were collected from 36 healthy Thoroughbreds and 12 Thoroughbreds with colitis. Serum from each horse suffering from colitis was collected daily until death or recovery. Collected sera were digested with trypsin. Peptides obtained from serum proteins were measured by Q-Exactive HF Orbitrap mass spectrometer. The identification and quantification of peptides were performed using Proteome Discoverer version 2.2. RESULTS: On day 1 of treatment, eight proteins in the colitis group were upregulated (P < .05, more than a twofold change) compared with the healthy group. Among the eight proteins, biliverdin reductase B was significantly upregulated (P < .05) in the non-survivor group (n = 5) compared with the survivor group (n = 7). On the last day of the treatment, haemoglobin subunit alpha, clusterin, glyceraldehyde-3-phosphate dehydrogenase, lipopolysaccharide-binding protein, and biliverdin reductase B showed significant increases (P < .05) in the non-survivor group. MAIN LIMITATIONS: The number of the identified proteins is limited due to the existence of abundant proteins. CONCLUSIONS: Measuring the changes of these proteins together may enable a potential prognosis or early diagnosis of horses suffering from colitis.