Advancements in microfluidic and electrophoretic techniques for stallion sperm isolation.

Context Equine reproductive technologies are crucial for overcoming challenges in natural fertilisation, particularly in sub-fertile stallions and breeding programs focused on genetic conservation and performance enhancement. Assisted reproductive technologies (ARTs), such as artificial insemination (AI), intracytoplasmic sperm injection (ICSI), and in vitro fertilisation (IVF), improve fertility outcomes and enable breeding across geographical distances. Aims This review examines sperm isolation techniques used in ART, evaluating their efficacy, limitations, and potential to enhance reproductive success in equine breeding. Methods Traditional sperm isolation methods, including sperm washing and single-layer centrifugation (SLC), are compared with emerging techniques such as microfluidic-based technologies and electrophoretic separation to assess their ability to improve sperm quality while minimising DNA damage. Key results While conventional methods are widely used, they present limitations, such as reduced motility, cost, and potential DNA damage. Novel approaches, including the VetMotl™ and Samson™, replicate natural sperm selection to enhance motility while preserving DNA integrity, and the electrophoretic sperm isolation device, Felix™, separates sperm based on surface charge and motility, benefiting cryopreserved samples. These innovations offer promising improvements in ART outcomes, though challenges remain, including high costs and limited sperm yields. Conclusions Emerging sperm isolation techniques have the potential to improve ART success, but further research is required to optimise these methods and validate their efficacy in fertility trials. Implications Advancements in sperm isolation could modernise equine reproductive practices by improving sperm quality and fertility outcomes, though accessibility and practical applications require continued investigation.