Isolation of high-quality stallion spermatozoa: a comparison of methods and their effects on sperm quality and yield, DNA damage, and functional competence by ICSI

Context Intracytoplasmic sperm injection (ICSI) is the primary method for equine in vitro embryo production, yet outcomes may be influenced by sperm selection techniques. Aims This study compared four isolation methods: EquiPure™, Vetmotl™, Felix™, and Samson™, to assess effects on sperm quality and embryo development. Methods Semen was collected from four fertile stallions, processed and evaluated for sperm motility, viability, DNA integrity, and yield of high-quality cells relative to input. Isolated samples were compared to non-isolated (centrifuged) samples. Spermatozoa were used for ICSI with in vitro–matured oocytes, and cleavage and blastocyst rates were recorded. Key results Compared with unfractionated samples, sperm quality (motility and viability) was improved in samples recovered with VetMotl, Felix and Samson while DNA integrity was improved in Vetmotl-isolated samples. When concentration of recovered cells was considered, Samson, EquiPure, and Felix yielded significantly more motile, viable, and DNA-intact spermatozoa than Vetmotl, showing the lowest recovery. Samson achieved the greatest recovery of progressively motile cells. Isolation with all methods supported in vitro embryo production; cleavage (51.2–69.4%) and blastocyst rates (20.5–24.5%) did not differ significantly between methods. Conclusions Several established and emerging methods for sperm isolation are effective in isolating high-quality spermatozoa suitable for use in embryo production by ICSI. VetMotl, Felix, and Samson performed comparably to conventional methods, supporting their clinical utility. Implications Isolation methods vary in efficiency, labour intensiveness and sperm selection stringency, with potential implications for sperm storage, low dose insemination and IVF. Further studies incorporating multiple stallions and embryo transfer outcomes are needed to confirm reproductive relevance.