Expert Q & A

Staying Ahead of Gene Doping in Equine Sport

By Adam Marcus

As long as horses have been used for sport, their handlers have looked for ways to give their mounts an unfair edge. In ancient Rome, the practice of giving chariot horses a drink akin to mead, called hydromel, to boost their performance during races was apparently so effective—and so frowned upon—those who were found guilty of administering the brew faced the death penalty. These days, the equivalent of hydromel doesn’t come in a bowl or trough, but likely is conjured up in a genetics laboratory, as researchers look for ways to modify the equine genome to maximize speed, muscle mass, recovery and other athletic characteristics.

We spoke recently with Teruaki Tozaki, PhD, manager of the genetic analysis section, Laboratory of Racing Chemistry in Tochigi, Japan, about the scope of gene doping in horses, the state-of-the-art for detecting cheating, and more. Dr. Tozaki is also a technical advisor in the Genetic Analysis Department.

MEV: What are the primary gene therapy techniques that could be misused for gene doping in horses, and how might they enhance athletic performance?

Dr. Tozaki: This approach includes transgene technology using plasmid vectors and adeno-associated virus (AAV) vectors. Transgene technology would be applied to genes that positively influence athletic performance. For example, the introduction of the IGF-1 gene could promote growth, while the use of erythropoietin may enhance oxygen-carrying capacity, thereby improving endurance.

Genome editing technology, particularly CRISPR (clustered regularly interspaced short palindromic repeats), also raises concerns. The application of genome editing in eggs or embryos results in the birth of genetically modified animals. This technology is used primarily to disrupt genes that have a negative regulatory function, as it facilitates gene knockouts. For instance, knocking out the MSTN gene, which inhibits muscle growth, may lead to increased muscle mass and the development of stronger horses. In Argentina, horses with MSTN gene edits have already been produced.

MEV: Which specific genes have been identified as potential targets for gene doping to improve equine performance, and what roles do they play in exercise adaptation?

Dr. Tozaki: The MSTN gene is one of the key genetic factors regulating muscle development. In the promoter region of this gene, some individuals carry an insertion sequence known as SINE, while others do not. Horses with this insertion exhibit reduced expression of the MSTN protein, greater muscle mass—primarily Type IIx fibers—and are better suited for short-distance racing. In contrast, horses without the insertion have higher expression of the protein, slightly lower muscle mass—predominantly Type I fibers—and are more suited for long-distance races.

Historically, horse races were typically run over longer distances, ranging from 3-5 km. However, in recent years, we have seen an increase in the number of races between 1 km and 1.6 km. As a result, the frequency of horses carrying the SINE insertion has increased, likely due to selective breeding favoring short-distance performance.

MEV: What are some direct and indirect detection methods available for identifying gene doping in horses, and what are their respective advantages and limitations?

Dr. Tozaki: When a gene doping agent is introduced as a transgene into the body of a postnatal animal, its detection typically is performed using quantitative polymerase chain reaction—real-time PCR or digital PCR. In such cases, the primary targets are plasmid or AAV vectors, which can be identified directly. In conventional doping tests for low-molecular-weight compounds, direct detection of the substance is the standard approach, making PCR the gold standard.

However, a drawback of the PCR method is that the number of target genes is limited. One method to address this issue is whole-genome sequencing.

On the other hand, false-positive results due to pseudogenes in the genome are a concern, and research is being conducted into countermeasures to this. In addition to transgenes, nucleic acid drugs are also being developed. These cannot be detected by PCR, so mass spectrometry is used. Indirect detection expands the possibilities for doping detection, and we are developing detection methods using miRNA as biomarkers.

MEV: What ethical concerns arise from the potential use of gene doping in equine sports, particularly regarding animal welfare and the integrity of competition?

Dr. Tozaki: Gene doping refers to the illegal use of gene therapy medicines or related gene therapy technologies to enhance performance. However, when approved by the appropriate regulatory bodies, gene therapy medicines that promote the health and well-being of horses could contribute positively to animal welfare. Conversely, the use of unapproved gene therapy medicines should be strictly prohibited to maintain fairness in competition. Currently, no gene therapy drugs for horses are commercially available, meaning their use would likely be banned by default.

Gene doping is not limited to the administration of gene-altering substances; it also includes the creation of genetically modified animals. This practice raises concerns not only regarding the welfare of horses, particularly the potential negative effects of genetic modifications, but also about biodiversity.

MEV: How can the concept of an equine biological passport be implemented to monitor and detect gene doping over time?

Dr. Tozaki: Biological passports are a crucial tool for preventing doping in sports. However, implementing this approach in horse racing and equestrian sports requires extensive clinical data collection from Thoroughbreds and various other breeds, making it challenging to administer effectively. For this reason, rather than relying on biological passports, it is more practical to focus on individual identification, systematic management, and the long-term storage of collected test samples. In this regard, close cooperation between horse racing authorities, studbook authorities, and laboratories is essential to ensuring fairness in competition.

MEV: What strategies should be employed to educate stakeholders, including veterinarians, trainers and the public, about the risks and ethical implications of gene doping in equine sports?

Dr. Tozaki: To effectively address gene doping, it is essential to promote the following 3 measures:
1. Clarifying regulations related to gene doping (FEI is set to update its rules this year).
2. Informing and educating stakeholders about the regulations, ensuring clear communication on the ethical and legal issues surrounding gene doping.
3. Implementing testing protocols to create a deterrent effect.
In addition, as various gene therapy technologies continue to evolve, regulatory frameworks must be continuously updated to reflect these advancements. MeV

For More Information
Tozaki T, Ohnuma A, Takasu M, et al. Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control. Gene Ther. 2021;28(3-4):199-205. https://pubmed.ncbi.nlm.nih.gov/32770095/

Furukawa R, Tozaki T, Kikuchi M, et al. A method for detecting gene doping in horse sports without DNA extraction. Drug Test Anal. 2025;17(3):427-435. https://doi.org/10.1002/dta.3745

Tozaki T, Ohnuma A, Kikuchi M, et al. Identification of processed pseudogenes in the genome of Thoroughbred horses: Possibility of gene-doping detection considering the presence of pseudogenes. Anim Genet. 2022;53(2):183-192.
https://onlinelibrary.wiley.com/doi/10.1111/age.13174

Kikuchi M, Ishige T, Minamijima Y, et al. Identification of potential miRNA biomarkers to detect hydrocortisone administration in horses. Int J Mol Sci. 2023;24(19):14515. doi: 10.3390/ijms241914515. https://pmc.ncbi.nlm.nih.gov/articles/PMC10572364/