Abstract #T327

# T327
Antioxidants improve membrane integrity and acrosome and sperm mitochondrial activity in ram sperm after cryopreservation.
Elenice A. Moraes*1, Wildelfrancys L. Souza1, Jonathan M. S. Costa1, James K. Graham2, 1Federal University of San Francisco Valley, Petrolina, PE, Brazil, 2Colorado State University, Fort Collins, CO.

There has been a renewed interest in the effects of oxidative damage to human sperm, as this damage to mitochondrial DNA and membrane architecture may explain the impaired fertility of the cryopreserved sperm. Different antioxidants have been tested to improve sperm quality but distinct and consistent beneficial effects are lacking. The objective was to determine if adding a combination different antioxidants improved ram sperm quality after cryopreservation. Thirty ejaculates, from 3 rams, were split and diluted into an egg-yolk-tris diluent containing different antioxidant treatments: control; 100 μM melatonin (MEL) plus 0.05% ascorbic acid (MEL+AA); 100 μM MEL plus 90 μL Trolox-C (MEL+TRO); 90 μL TRO plus 0.05% AA (TRO+AA); and 100 μM MEL plus 0.05% AA plus 90 μL of TRO (MEL+AA+TRO); to final concentration 200 × 106 sperm/mL. The samples were cooled to 5°C/2 h, packaged into 0.5-mL straws, and frozen in static liquid nitrogen vapor for 15 min before being plunged into liquid nitrogen. Straws were thawed (37°C/30s). The motility was determine using CASA, and to plasma membrane integrity the samples were stained with 2 L of Hoechst 33342 and 2 μL PI and incubated at 37°C/8min. Acrosomal integrity was determined, visually, after staining the sperm with 50 μL FITC-PNA at 37°C/20min. The sperm mitochondrial function was assessed using Rhodamine and PI (samples were incubated at 37°C/8min with at 2 μL Rhodamine and 2 μL PI) and the samples assessed for the proportion of viable spermatozoa with high mitochondrial activity. Variables were determined by ANOVA using a Tukey test. Sperm treated with MEL+AA (29.1%) and MEL+AA+TRO (28.9%) maintained higher percentages of cells with intact plasma membranes after thawing (P < 0.05) than other treatments. The antioxidant combination (MEL+AA+TRO) resulted in the highest percentages of sperm with intact acrosomes (84.5%) and mitochondrial activity (96.4%) compared with other treatments (P < 0.05). All antioxidant treatments exhibited higher motility (61.4% versus 53%), acrosome integrity (78.1% versus 68.2%) and mitochondrial activity (92.7% versus 88.6%), than control (P < 0.05).Therefore, adding a combination of MEL+AA+TRO to ram sperm improved cell cryosurvival. Supported by FACEPE/CAPES.

Key Words: melatonin, Trolox-C