Extender osmolality and sugar supplementation exert a complex effect on the cryopreservation of Iberian red deer ( Cervus elaphus hispanicus) epididymal spermatozoa

We have carried out two experiments to study the cryobiology of red deer epididymal spermatozoa and to improve freezing extenders: (1) effect of extender (Tris-citrate–fructose) osmolality (300–600 mOsm/kg), and (2) effect of sugar (0.4 M) supplementation to the extender (no sugar, glucose, fructose, mannose, sucrose, maltose, threalose and raffinose). Sperm quality was assessed pre-freezing, post-thawing, and after 2 h at 37 °C post-thawing: sperm motility index (SMI), acrosome integrity and membrane integrity (HOS test) were assessed subjectively; mitochondrial activity (JC-1) and membrane stability (merocyanine 540) were assessed by flow cytometry. In experiment 2, DNA status was assessed using acridine orange and flow cytometry. To find an optimal osmolality, we fitted the data to a quadratic curve. Four hundred Osmolal per kilogram rendered better results pre-freezing and post-thawing. However, post-thawing viability and most parameters after 2 h incubation fitted a linear model. Osmolalities above 425 mOsm/kg were deleterious ( P < 0.05). In experiment 2, fructose had a positive effect respect to control after 2 h of incubation at 37 °C post-thawing. Di and trisaccharides were deleterious. Trehalose showed impaired DNA status after 2 h incubation. In conclusion, the osmolality of the extenders should be around 400 mOsm/kg, although the change from quadratic to lineal may indicate a complex effect that must be further studied. Monosaccharides may enhance red deer epididymal sperm cryopreservation, especially fructose, whereas di and trisaccharides may not be adequate.