Influence of cooling rate on the ability of frozen–thawed sperm to bind to heterologous zona pellucida, as assessed by competitive in vitro binding assays in the ocelot (Leopardus pardalis) and tigrina (Leopardus tigrinus)

We evaluated the influence of two cooling rates (from 25 to 5 °C) on post-thaw function of frozen sperm in ocelots (Leopardus pardalis; n = 3 males) and tigrinas (Leopardus tigrinus; n = 4 males). Seven normospermic (>70% normal sperm) electroejaculates from each species were diluted with a 4% glycerol freezing medium, divided into two aliquots, and assigned to one of two cooling rates: fast or slow (0.7 or 0.16 °C/min, respectively). Sperm motility index (SMI) and percentage of sperm with an intact acrosome were assessed before freezing and after thawing, and the ability of sperm to bind to the zona pellucida of IVM domestic cat oocytes were assessed in a competitive in vitro sperm-binding assay. Regardless of the cooling rate, frozen–thawed sperm from both species exhibited a SMI of 50; ∼20 and ∼32% of post-thaw sperm had an intact acrosome in ocelots and tigrinas, respectively (P < 0.05). The mean (±S.E.M.) number of sperm bound per oocyte was higher for fast-cooled (8.5 ± 1.3) than slow-cooled (2.5 ± 0.3; P < 0.01) ocelot sperm. In contrast, more tigrina sperm bound to domestic cat oocytes when cooled slowly versus quickly (5.8 ± 0.9 versus 2.7 ± 0.4, P < 0.05). In conclusion, cryopreservation decreased sperm function in both species, and the oocyte-binding assay was the most efficient method to detect functional differences in post-thaw sperm.