During freeze-preservation of high-viability ejaculates of horse semen the duration of the equilibration time for nucleated straws (achieved at -7 ⁰C following induced nucleation and during controlled, slow cooling to -60 ⁰C) has little impact on viability, measured using propidium iodide staining to indicate cells that have lost osmotic competence.  Further, relatively high viability is retained if direct plunging into liquid nitrogen (LN) replaces the controlled protocol, and the sample can withstand several cycles of repeated freezing. The data suggests that populations of spermatozoa with a high viability have a large cohort of extremely durable, freeze-tolerant cells. Preliminary observations suggest that populations with low overall viability may not behave, qualitatively, in the same way, suggesting fundamental cellular differences. The cryobiology underlying these observations is discussed and new approaches for successful cryopreservation of semen of low viability are considered.

Keywords

Equine spermatozoa; Cryopreservation; Nucleation; Osmotic stress; Repeated freezing