Osmotic properties of spermatozoa from felids producing different proportions of pleiomorphisms: influence of adding and removing cryoprotectant.

The spermatozoon of felids (cats) survives cryopreservation inconsistently. Using ejaculates from three species (domestic cat [normospermic versus teratospermic], the normospermic serval and the teratospermic clouded leopard), this study (1) determined the influence of adding and removing two permeating cryoprotectants (glycerol and dimethylsulfoxide) and (2) assessed the impact of one-step versus multi-step cryoprotectant removal on sperm motility and membrane integrity. Spermatozoa were exposed in a single step to various anisotonic solutions or to 1M solutions of glycerol or dimethylsulfoxide. In both cases, sperm then were returned to near isotonic conditions in a single or multi-step with de-ionized water, Ham's F10 medium or saline. Percentage of sperm motility was measured subjectively, and plasma membrane integrity was assessed using a dual fluorescent stain and flow cytometry. Sperm motility was more sensitive to anisotonic conditions than membrane integrity. Rapid dilution into various test solutions and removal of cryoprotectant with de-ionized water reduced (P<0.01) sperm motility compared to control spermatozoa maintained in Ham's F10. Exposing sperm from all species to a 1M solution of either cryoprotectant resulted in >85% spermatozoa retaining intact membranes. However, return to isotonicity with de-ionized water in a single step or multiple steps always caused severe plasma membrane disruption. In contrast, sperm motility and membrane integrity in all species and populations remained unaffected (P>0.05) when spermatozoa were returned to isotonicity in multiple steps with Ham's F10 medium or 0.9% sodium chloride. Results demonstrate that: (1) felid spermatozoa are resistant to hypertonic stress; (2) sperm motility is more sensitive to changes in osmolality than membrane integrity; and (3) removal of cryoprotectant in multiple steps with an isotonic solution minimizes loss of sperm motility and membrane disruption in both normospermic and teratospermic males.