Assisted reproductive techniques (ARTs) were originally developed to overcome fertility challenges. ARTs encompass techniques associated with the in vitro handling, manipulation and production of sperm, oocyte (male and female gamete) and embryo, typically for the purpose of establishing a pregnancy. In the equine industry, ARTs were initially used in sub-fertile mares and stallions, but their increasing possibilities and economic benefits have driven their widespread adoption.
An introduction to ARTs
Artificial insemination (AI) is by far the most commonly used ART in horses. The routine use of cooled/transported or frozen semen has facilitated the incorporation of valuable genetics that would otherwise be geographically inaccessible. The benefits of frozen semen extend beyond convenience and include allowing stallions to maintain their sports careers without reproductive interruptions, preserving genetics post-mortem and enabling international semen transport.
For mares, their long gestational period limits reproductive output to one foal per year. This constraint has driven the popularity of embryo collection and transfer, also known as in vivo embryo production (IVV). This ART enables genetically valuable mares to produce multiple offspring per season without compromising their sports careers.
In vitro embryo production
Beyond these well-established ARTs, advancements in technology and improved success rates in recent years have further increased interest in in vitro embryo production (IVP), which often yields higher embryo production rates than traditional embryo collection (Malin et al., 2024).
The main technique used for in vitro embryo production is transvaginal aspiration of oocytes (TVA)/intracytoplasmic sperm injection (ICSI) (Stout, 2020). TVA, also known as ovum pick-up (OPU), involves the collection of oocytes directly from the ovary of a standing donor mare. Since mares are predominantly monotocous (producing one ovulation per cycle) and there are no commercially available superovulation protocols, immature oocytes are typically aspirated from follicles at any stage of the heat cycle.
Collected immature horse oocytes can be held or transported in commercially available embryo-holding media at approximately 20°C for up to 24 hours before maturation without detrimental effects
IVP requires a highly specialised facility and trained personnel, limiting its widespread adoption. However, a significant advantage is that the collected immature horse oocytes can be held or transported in commercially available embryo-holding media at approximately 20°C for up to 24 hours before maturation without detrimental effects. This capability has facilitated overnight transport of oocytes to specialised IVP laboratories, contributing to the increasing popularity of TVA, as more clinics now offer this service.
In vitro fertilisation techniques
In vitro fertilisation can occur in two ways:
- Intracytoplasmic sperm injection (ICSI): a single sperm cell is directly injected into a matured oocyte
- Conventional in vitro fertilisation (IVF): sperm is capacitated and co-incubated with matured oocytes, allowing fertilisation to occur naturally
In horses, ICSI is the routine technique, due to the historical failure of conventional IVF.
One of the advantages of ICSI is that since a single sperm cell is injected directly into each mature oocyte, it requires only a minimal amount of semen. This allows for the use of a small cut from a frozen semen straw, which enables multiple uses from a single straw – an attractive feature when semen availability is a concern. However, beyond the necessary equipment and facilities required for both types of IVP, ICSI specifically requires a specialised micromanipulator and an embryologist proficient in its use, limiting this technique to only a few facilities worldwide.
Unlike ICSI, conventional IVF does not require personnel trained in microinjection – a technique with a steep learning curve. For this reason, researchers in the field of equine reproduction have continuously sought to develop reliable IVF methods for horses. However, only a few foals were produced using IVF and there was no real repeatability in their research.
Innovative protocols for IVF in 2025
Considering the high embryo production rate of ICSI and its proven success in producing live foals, IVF still needed to demonstrate its efficiency
In 2022, a new protocol involving prolonged sperm capacitation using fresh semen was published, resulting in the successful birth of live foals (Felix et al., 2022). This publication marked a new beginning, opening the door for new perspectives in equine ART. However, the protocol’s reliance on fresh semen limited its efficiency in a commercial setting, as the most sought-after stallions are typically only available via frozen semen. Furthermore, considering the high embryo production rate of ICSI and its proven success in producing live foals, IVF still needed to demonstrate its efficiency.
With these premises, Martin-Pelaez et al. (2025) developed a protocol for IVF using frozen–thawed sperm and conducted an early embryonic development comparison between ICSI and IVF embryos. Using frozen semen, the authors found that both techniques can generate a similar number of embryos, with a blastocyst rate of 41.1 percent for IVF and 41.8 percent for ICSI. The ability to use frozen semen broadens the technique’s commercial applicability, making it a viable option for the equine industry.
Interestingly, IVF embryos developed faster than their ICSI counterparts and were also larger in diameter. Altogether, this potentially suggests a more efficient early embryonic developmental competency and higher-quality embryos in the IVF group. However, further evaluation of pregnancy outcomes is necessary to determine their full potential. Overall, this is a promising step forward, demonstrating that IVF has the potential to at least match current ICSI protocols in terms of embryo production.
What needs to change to move forward with equine IVF in 2025?
Despite these exciting advancements, several factors must be considered before the widespread adoption of IVF.
Firstly, there is a significant stallion effect on embryo production. Some stallions tested were incapable of producing embryos, while others showed high variability between batches of frozen semen. This underscores the necessity of high-quality sperm samples, as low-motility sperm do not survive the prolonged capacitation period required for successful fertilisation. The authors anticipate that stallion testing will become a required step before offering IVF embryo production commercially.
Secondly, the sperm quantity required for IVF versus ICSI is vastly different. As mentioned earlier, ICSI requires only a few sperm cells per session (ICSI cycle), whereas the current IVF protocol necessitates a sperm concentration of 1 million/ml for fertilisation (IVF cycle). This could be a limitation for stallions with restricted semen availability.
Thirdly, while IVF is often perceived as requiring less intensive and prolonged training than ICSI, making it more accessible, the reality is that IVP extends beyond fertilisation. It involves critical steps such as oocyte maturation, embryo culture and blastocyst cryopreservation, all of which demand a highly specialised laboratory with well-established protocols. Underestimating these requirements before setting up an IVP facility can lead to significant challenges, both in terms of operational efficiency and financial investment (Scoggin et al., 2024).
Conclusion
In vitro fertilisation with frozen–thawed semen is now a viable option, demonstrating embryo yields comparable to intracytoplasmic sperm injection for in vitro embryo production
Overall, IVF with frozen–thawed semen is now a viable option, demonstrating embryo yields comparable to ICSI for IVP. However, further research is needed to evaluate its pregnancy potential, as well as stallion- and mare-specific factors that influence fertilisation using this method.
While additional studies are required before widespread commercial application, significant advancements in this field can be expected in the coming years.
