38 Sperm selection improves the quality of bovine epididymal spermatozoa cryopreserved by slow and ultrarapid freezing

This study aimed to assess the effect of sperm selection before cryopreservation on bovine epididymal sperm quality after freezing and thawing. Fifteen testicular pairs were used to recover spermatozoa samples by retrograde flushing of the cauda epididymis with 1 mL of extender. Sperm samples were split into two aliquots: (1) for slow freezing (SF) and (2) for ultrarapid freezing (UF). Half of each aliquot was selected by centrifugation (300 × g for 20 min) by density gradients (200 µL of 40% and 80% Percoll). Samples were diluted in TSG (tris, citric acid, glucose) + egg yolk (6%, vol/vol) with glycerol (5%) or sucrose (100 mM), cooled at 5°C × 30 (UF) or 120 (SF) min. For UF, drops of ~30 µL of the vitrification solution were dropped into liquid nitrogen, and warmed in a heating platform at 65°C. For SF, samples, previously loaded in 0.25 mL straws, were frozen by placing the straws in nitrogen vapors for 10 min prior plunging them into liquid nitrogen, and thawed in a water bath at 37°C for 30 s. Sperm motility and head morphometry were assessed by CASA system. Plasma and acrosome membrane status were assessed with propidium iodide and fluorescein isothiocyanate conjugated peanut agglutinin. The DNA integrity was evaluated by acridine orange. Data were analysed by GLM and LSM of SAS. Mean values of the kinetic variables and status of plasma and acrosome membranes are shown in Table 1. Proportion of epididymal spermatozoa with intact plasma and acrosomal membranes was significantly greater in the selected sperm samples for both freezing methods. Neither sperm head dimensions nor DNA integrity were affected by sperm selection or freezing method. Collectively, these results indicate that selecting sperm samples prior cryopreservation is a valid method for improving sperm quality and viability after freezing and thawing.