Sperm viability in the black-footed ferret (Mustela nigripes) is influenced by seminal and medium osmolality

Fundamental knowledge of spermatozoa cryobiology can assist with optimizing cryopreservation protocols needed for genetic management of the endangered black-footed ferret. Objectives were to characterize semen osmolality and assess the influence of two media at various osmolalities on sperm viability. We examined the influence of Ham’s F10 +Hepes medium (H) at 270, 400, 500 or 700mOsm (adjusted with sucrose, a nonpermeating cryoprotectant) and TEST Yolk Buffer (TYB) with 0% (300mOsm) versus 4% (900mOsm) glycerol (a permeating cryoprotectant). Electroejaculates (n=16) were assessed for osmolality using a vapor pressure osmometer. For media comparison, semen (n=5) was collected in TYB 0%, split into six aliquots, and diluted in H270, H400, H500, H700, and TYB 0% or TYB 4%. Each sample was centrifuged (300g, 8min), resuspended in respective medium, and maintained at 37°C for 3h. Sperm motility and forward progression were monitored every 30min for 3h post-washing. Acrosomal integrity was monitored at 0 and 60min post-washing. Results demonstrated that black-footed ferret semen has a comparatively high osmolality (mean±SEM, 513.1±32.6mOsm; range, 366–791mOsm). Ferret spermatozoa were sensitive to hyperosmotic stress. Specifically, sperm motility was more susceptible (P<0.01) to hyperosmotic conditions than acrosomal integrity, and neither were influenced (P>0.05) by hypotonic solutions. Exposure to TYB 4% glycerol retained more (P<0.01) sperm motility than a hyperosmotic Ham’s (700mOsm). These findings will guide the eventual development of assisted breeding with cryopreserved sperm contributing to genetic management of this rare species.