A Simple Microaspiration Technique for Isolating Somatic Cells from Cryopreserved Equine Semen as Nuclear Donors for Cloning

Semen is a complex fluid that, in addition to spermatozoa, contains other cell populations, including immune cells, immature male germ cells, epithelial cells, and fibroblasts. These cells share the diploid condition, making them suitable candidates as nuclear donors for somatic cell nuclear transfer (SCNT) cloning. The generation of viable embryos and offspring has been demonstrated using these cells. Effective methods for isolating them from semen include centrifugation and osmotic gradient techniques; however, prolonged in vitro culture periods are necessary to establish primary cultures from these isolated cells. Furthermore, the samples that were obtained contained a high load of spermatozoa, which interferes with the establishment and maintenance of in vitro cultures. To date, primary cultures have only been successfully established from fresh semen samples, while attempts using cryopreserved semen have consistently failed. This limitation significantly restricts the potential to generate clones from cryopreserved semen samples. The present study proposes a simple microaspiration-based methodology for isolating somatic cells from cryopreserved equine semen. This approach allows for the rapid retrieval of a sufficient number of cells for immediate use in SCNT cloning procedures.