The post-thaw quality of ram sperm held for 0 to 48 h at 5 °C prior to cryopreservation

The effects of holding diluted ram semen at 5 °C for up to 48 h prior to cryopreservation were investigated. Semen from six rams was collected by electro-ejaculation in the autumn and again from six different rams in the spring. The sperm concentration and motility were determined using spectrophotometry and computerized automated semen analysis, respectively. Samples were diluted at 23 °C to 400 × 106 cells/ml in a one-step Tris–egg yolk–glycerol (5%, v/v) media, cooled to 5 °C over 2 h and maintained at 5 °C for the duration of the experiments. Aliquots were loaded into 0.5 ml French straws at 0, 24 or 48 h after cooling, frozen in liquid nitrogen vapor for 12–13 min, 4.5 cm above the liquid nitrogen, and plunged into liquid nitrogen for storage. After thawing, autumn samples frozen after 0, 24, or 48 h of storage exhibited similar percentages of motility (29, 31, 36%, respectively), progressively motility (16, 15, 17%, respectively), plasma membrane integrity (28, 35, 29%, respectively) and live acrosome-reacted cells (0.4, 0.6, 0.8%, respectively; P > 0.05). In addition, the quantity of sperm that bound to hen's egg perivitelline membranes after being held at 5 °C for 0, 24, or 48 h was not significantly different when the values were expressed as means of the quantity of sperm (155, 177, 106 sperm, respectively) or as the proportion of sperm inseminated (0.39, 0.49, 0.34, respectively; P > 0.05). Likewise, ram sperm collected in the spring and frozen at 0, 24 and 48 h after cooling had similar (P > 0.05) total motility (21, 25, 20%, respectively), progressive motility (14, 15, 11%, respectively), plasma membrane integrity (26, 33, 31%, respectively) and live acrosome-reacted cells (3.7, 3.5, 3.2%, respectively; P > 0.05). The 0 h holding time had significantly less sperm bound to a hen's egg perivitelline membrane compared to the 48 h holding time (250 and 470 sperm, respectively) although the 24 h holding time was not different from the 0 or 48 h holding time (281 sperm; P < 0.05) but analysis of the proportion of the total sperm inseminated resulted in no significant differences observed (P > 0.05). These results indicate that ram sperm can be held at 5 °C for up to 48 h prior to freezing with no injurious effects on motility, membrane integrity, or fertilizing potential as indicated by membrane binding ability.