Researchers from the University of Florida Department
of Animal Sciences have reported
progress in equine reproduction,
demonstrating that frozen-thawed stallion sperm may be more effective for in
vitro fertilisation (IVF) than fresh or chilled semen. The findings, published
in the journal Reproductive Biology, could have significant implications
for the performance horse industry.
In horse breeding, IVF has historically lagged behind its
success in other species such as cattle. One major challenge has been the
difficulty of achieving proper sperm function outside the body. In particular,
stallion sperm require a process known as capacitation before they
can penetrate and fertilise an egg. This involves a series of physical and
biochemical changes that are highly sensitive to environmental conditions such
as temperature and time.
The need for improved IVF techniques is especially relevant
in the performance horse sector. Highly valuable mares are often selected for breeding,
but not all are able to safely conceive or carry a pregnancy. Certain
conditions, including uterine disease, prior foaling injuries, or lameness, may
increase the risk associated with pregnancy. Additionally, some mares may be
performing at their optimal level and cannot be withdrawn from competition for
breeding purposes.
In such cases, producing an embryo via IVF and transferring
it into a surrogate mare offers a safer alternative, protecting both the donor
mare and the developing foal.
To better understand how to achieve successful
fertilisation, the research team investigated how different types of semen - fresh,
chilled, and frozen-thawed - respond under capacitating conditions. They also
compared various sperm selection methods, including Percoll gradients, Swim-Up
techniques, and microfluidic systems*. These approaches were assessed based on
key indicators such as sperm motility, acrosome integrity, and biochemical
markers like tyrosine phosphorylation.
The results revealed that temperature played a critical
role. Incubating sperm at body temperature (38°C) reduced motility, whereas
maintaining sperm at ambient temperature allowed optimal capacitation within
approximately four hours. At this time point, sperm also showed the highest
level of acrosome integrity, an important factor for successful fertilisation.
The research team found that frozen-thawed sperm performed
better than fresh or chilled samples in the IVF setting. Although freezing and
thawing can place mild stress on sperm cells, this stress appears to trigger
changes that promote capacitation. As a result, frozen sperm were able to
function effectively with fewer preparatory steps.
Further experiments confirmed fertilisation success through
the observation of pro-nuclear formation and early embryo development. Notably,
microfluidic sperm selection produced superior results compared to traditional
Swim-Up methods, improving sperm quality and fertilisation potential.
Beyond the biological findings, the use of frozen sperm
offers practical advantages. Unlike chilled semen, which must be used within a
short timeframe, frozen semen can be stored indefinitely and thawed when
needed. This flexibility allows veterinarians and breeders to better coordinate
the timing of egg collection and fertilisation, making the IVF process more
efficient and accessible.
This research represents a significant step forward after
more than three decades of limited progress in equine IVF. By identifying
conditions that support effective capacitation and demonstrating the advantages
of frozen sperm, the study provides a foundation for improving embryo
production in horses. For the equine industry, these advances could expand
breeding options while prioritising the health and welfare of valuable mares.
For more details, see:
Joao D. de Agostini Losano, Jillian E. Guertin, Maura S.
McGraw, Lana Katz, Vilceu Bordignon, Justin W. Callaham, Jose H.F. Pontes,
Perla Fleury, Marc Maserati, Carly Turner, Bradford W. Daigneault,
Temporal characterization of conditions that promote
functional capacitation of stallion sperm,
Reproductive Biology (2026) Vol 26, 2,101201,
https://doi.org/10.1016/j.repbio.2026.101201
*Percoll gradient centrifugation is a method that
separates sperm based on their density. A liquid medium is layered in tubes at
different concentrations, and the semen sample is placed on top. When the tube
is spun in a centrifuge, sperm cells move through the layers depending on their
density and quality. The most viable sperm - those that are motile and
structurally normal - tend to settle into a specific layer, while debris, dead
sperm, and less functional cells remain elsewhere. This method is widely used
because it produces a relatively clean and concentrated sample of high-quality
sperm.
Swim-Up technique relies on sperm motility rather than
density. In this approach, a semen sample is placed at the bottom of a tube
with a layer of culture medium above it. Over time, the most motile sperm
actively swim upward into the medium. These sperm are then collected from the
top layer. This technique mimics a natural selection process, as only the
strongest swimmers are chosen. However, it can result in lower sperm numbers
and may not always select for the best overall sperm quality, especially if
motility is reduced.
Microfluidic sperm selection systems represent a more
modern and precise approach. These systems, such as microfluidic sperm
selection, use tiny channels that mimic aspects of the female reproductive
tract. Sperm are introduced into a device where they must navigate through
narrow pathways and fluid flows. Only sperm with good motility, proper
morphology, and functional membranes can successfully pass through the system.
This method reduces mechanical stress on the sperm compared to centrifugation
and may better preserve their physiological state.