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.

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.

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.

Monitoring the positive conversion of anti-erythrocyte antibodies in blood transfusion donor horses.

To confirm the positive conversion of antibodies against erythrocyte antigens in horses, possible blood transfusion donor horses selected from draft horse populations were periodically monitored with an indirect antiglobulin (Coombs) test for approximately 3 years. In this study, 19 horses (16 females and 3 males) were investigated, and five mares showed alloantibodies during the monitoring period. Four mares were typically pregnant when positive conversion was detected, whereas no particular cause of conversion could be observed for one mare based on its clinical records. In the analyzed horses, most positive conversions were possibly due to pregnancy, as conversion occurred more often during this period than after parturition. Pregnancy is considered a key event for positive conversion. Additionally, in cases in which unknown causative sensitization is confirmed, continuous monitoring with a test to detect antibodies should be performed, even if the possible donor is selected and maintained.

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 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.

Efficient simultaneous double DNA knock-in in murine embryonic stem cells by CRISPR/Cas9 ribonucleoprotein-mediated circular plasmid targeting for generating gene-manipulated mice.

Gene targeting of embryonic stem (ES) cells followed by chimera production has been conventionally used for developing gene-manipulated mice. Although direct knock-in (KI) using murine zygote via CRISPR/Cas9-mediated genome editing has been reported, ES cell targeting still has merits, e.g., high throughput work can be performed in vitro. In this study, we first compared the KI efficiency of mouse ES cells with CRISPR/Cas9 expression vector and ribonucleoprotein (RNP), and confirmed that KI efficiency was significantly increased by using RNP. Using CRISPR/Cas9 RNP and circular plasmid with homologous arms as a targeting vector, knock-in within ES cell clones could be obtained efficiently without drug selection, thus potentially shortening the vector construction or cell culture period. Moreover, by incorporating a drug-resistant cassette into the targeting vectors, double DNA KI can be simultaneously achieved at high efficiency by a single electroporation. This technique will help to facilitate the production of genetically modified mouse models that are fundamental for exploring topics related to human and mammalian biology.

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.

Identification of processed pseudogenes in the genome of Thoroughbred horses: Possibility of gene-doping detection considering the presence of pseudogenes.

Processed pseudogenes, also known as retrocopy genes, are copies of messenger RNAs that have been reverse transcribed into DNA and inserted into the genome. In this study, we identified 62 processed pseudogene candidates as intron-less genes from whole-genome sequencing (WGS) data of Thoroughbred horses using delly structural variation software. The 62 processed pseudogene candidates were confirmed by PCR amplification of intron-less products. A total of 11 processed pseudogenes were confirmed in the genome of all 23 analysed horses, whereas three processed pseudogenes with structures of ATP11B, DPH3 and RPL17 were detected in only one of 115 horses by PCR amplification of intron-less products. Currently, most of the gene doping tests proposed in human and horse sports are adapted PCR-based methods using hydrolysis probes to detect exon/exon junctions in transgenes because the operation is simple and economical. However, when the pseudogene is present in the host genome, the PCR-based methods may have a potential risk of detecting false positives. In this study, because processed pseudogenes that exist less frequently in the horse genome may affect PCR-based transgene detection in gene-doping tests, we propose and demonstrate that PCR amplification and sequencing using primers designed on transgene and promotors and/or polyadenylation signal for gene expression are useful for gene-doping detection as an additional confirmatory test to prevent false positives.

Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control.

Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected low-copy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene-doping detection.

Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports.

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 104 (standard deviation: 4.34 × 103 ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control.

Rare and common variant discovery by whole-genome sequencing of 101 Thoroughbred racehorses.

The Thoroughbred breed was formed by crossing Oriental horse breeds and British native horses and is currently used in horseracing worldwide. In this study, we constructed a single-nucleotide variant (SNV) database using data from 101 Thoroughbred racehorses. Whole genome sequencing (WGS) revealed 11,570,312 and 602,756 SNVs in autosomal (1-31) and X chromosomes, respectively, yielding a total of 12,173,068 SNVs. About 6.9% of identified SNVs were rare variants observed only in one allele in 101 horses. The number of SNVs detected in individual horses ranged from 4.8 to 5.3 million. Individual horses had a maximum of 25,554 rare variants; several of these were functional variants, such as non-synonymous substitutions, start-gained, start-lost, stop-gained, and stop-lost variants. Therefore, these rare variants may affect differences in traits and phenotypes among individuals. When observing the distribution of rare variants among horses, one breeding stallion had a smaller number of rare variants compared to other horses, suggesting that the frequency of rare variants in the Japanese Thoroughbred population increases through breeding. In addition, our variant database may provide useful basic information for industrial applications, such as the detection of genetically modified racehorses in gene-doping control and pedigree-registration of racehorses using SNVs as markers.

Robustness of digital PCR and real-time PCR against inhibitors in transgene detection for gene doping control in equestrian sports.

Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time PCR against gene doping. Digital PCR amplification was significantly inhibited by high concentrations of proteinase K (more than 0.1 µg/µl), ethylenediaminetetraacetic acid (more than 5 nmol/µl), and heparin (more than 0.05 unit/µl) but not by ethanol or genomic DNA. In addition, phenol affected droplet formation in the digital PCR amplification process. Real-time PCR amplification was inhibited by high concentrations of phenol (more than 1% v/v), proteinase K (more than 0.001 µg/µl), ethylenediaminetetraacetic acid (more than 1 nmol/µl), heparin (more than 0.005 unit/µl), and genomic DNA (more than 51.9 ng/µl) but not by ethanol. Although both PCR systems were inhibited by nearly the same substances, digital PCR was more robust than real-time PCR against the inhibitors. We believe that our findings are important for the development of better methods for transgene detection and prevention of false negative results in gene doping testing.

Robustness of Digital PCR and Real-Time PCR in Transgene Detection for Gene-Doping Control.

Gene doping is banned in human sports, horseracing, and equestrian sports. One possible form of gene doping is to administer exogenous genes, called transgenes. Several transgene detection methods based on quantitative PCR have been developed. In this study, we investigated the robustness of digital PCR and real-time PCR in transgene detection using primers and probes that matched (P-true) or incompletely matched (P-false) the template DNA. Fluorescence intensity was significantly reduced when substituted probes were used compared to that using the matched probe in both digital and real-time PCR assays. Digital PCR yielded a similar copy number regardless of the probe (P-true: 1230.7, P-false: 1229.7), whereas real-time PCR revealed a decrease in sensitivity based on Cq values (P-true: 23.5, P-false: 29.7). When substituted primers were used, the detected copy number decreased in the digital PCR assay, and the Cq value in real-time PCR was much higher. Interestingly, digital PCR copy numbers improved by performing PCR at a low annealing temperature, even if a substituted probe was used. Thus, when primer and probe sequences did not completely match the template transgene, digital PCR was relatively robust, but real-time PCR was less sensitive. Although PCR specificity may be reduced, PCR sensitivity can be improved by lowering the annealing temperature. If the target sequence is substituted to escape doping detection, it may be desirable to set the annealing temperature lower and use a more robust method, such as digital PCR, to increase the detection of positive cases, which will also result in fewer false-negative results.

Investigation of erythrocyte antigen frequencies in draft horse populations in Japan to assess blood donor suitability.

Erythrocyte alloantigen frequencies of draft horses in Japan were investigated to assess blood donor suitability for transfusion. Here, 148 Japanese draft, 69 Percheron, and 65 Breton horses were blood-typed and subjected to an indirect antiglobulin test. Regarding the major immunogenic factors, the rates of Aa- and Qa-negative horses ranged from 0.35 to 0.49 and from 0.82 to 1.00, respectively. The rate of alloantibody-positive horses ranged from 0.12 to 0.35. Although the prevalence of alloantibodies in these horses was higher than that expected naturally, the rates of Aa- and Qa-negative horses were higher than those of some breeds reported previously. The current draft horse population could provide potential candidates for donors, and the obtained information may contribute to the selection of a safe donor for transfusion.

Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests.

One method of gene doping in horseracing is administering of exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs for reference materials (RMs) and/or positive template controls (PTCs) are necessary for simultaneous analysis of multiple transgene targets. In this study, we designed and developed a novel RM for simultaneously detecting multiple targets via microfluidic quantitative PCR (MFQPCR). Twelve equine genes were selected as targets in this study. A sequence region including primers and probes for quantitative PCR was designed, and a 10 bp sequence was inserted to allow the RM to be distinguished from the original transgene sequences. The sequences of individual detection sites were then connected for 12 genes and cloned into a single plasmid vector. We performed fragment size analysis to distinguish between the PCR products of the original transgene sequence and those of the RM, enabling identification of RM contamination. PTCs diluted to 10,000, 1,000, 100, and 10 copies/µl with horse genomic DNA from RM were stably stored at 4°C for 1 year. As digital PCR enabled absolute quantification, the designed substances can serve as an RM. These findings indicate that the RM design and storage conditions were suitable for gene doping tests using MFQPCR.

Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control.

Gene doping has raised concerns in human and equestrian sports and the horseracing industry. There are two possible types of gene doping in the sports and racing industry: (1) administration of a gene-doping substance to postnatal animals and (2) generation of genetically engineered animals by modifying eggs. In this study, we aimed to identify genetically engineered animals by whole-genome resequencing (WGR) for gene-doping control. Transgenic cell lines, in which the erythropoietin gene (EPO) cDNA form was inserted into the genome of horse fibroblasts, were constructed as a model of genetically modified horse. Genome-wide screening of non-targeted transgenes was performed to find structural variation using DELLY based on split-read and paired-end algorithms and Control-FREEC based on read-depth algorithm. We detected the EPO transgene as an intron deletion in the WGR data by the split-read algorithm of DELLY. In addition, single-nucleotide polymorphisms and insertions/deletions artificially introduced in the EPO transgene were identified by WGR. Therefore, genome-wide screening using WGR can contribute to gene-doping control even if the targets are unknown. This is the first study to detect transgenes as intron deletions for gene-doping detection.