PS5-10. Effects of Lycopene Supplementation to Semen Extender on the Quality of Cooled and Frozen-thawed Ram Spermatozoa.

Abstract Ram sperm quality has been shown to significantly decrease following 3-5 days of liquid storage and during cryopreservation, primarily due to excessive production of reactive oxygen species (ROS). Lycopene, a carotenoid red pigment, mostly found in tomatoes and watermelon has100 times higher free radicals scavenging ability than other antioxidants. Our aim was to evaluate the effect of supplementing semen extender with lycopene on the sperm quality parameters of ram spermatozoa after cooling and freezing. Semen samples collected from 5 mature Katahdin rams once/week for 3 weeks using electroejaculation were pooled and diluted to a final concentration of 800 × 106 sperm/ml in Andromed® extender supplemented with 0 (control), 10, 20, and 30 µM lycopene. Diluted semen was then cooled at 4 °C for 72 h. Moreover, diluted semen was cooled at 4 °C for 3 h then frozen in 0.25 ml Frensh straws in a programmable freezer (MiniDigitcool, IMV®, USA). Straws were then plunged into LN2 before being thawed in water bath at 37 °C for 30 sec. Sperm motility and kinematics were evaluated using CASA at 4, 24, 48, and 72 h post-cooling and post-thawing. While post-cooling, viability, membrane, and acrosome integrity were evaluated using eosin-nigrosin stain and hypo-osmotic swelling (HOS) test, and FITC-PNA stain, respectively. Data were analyzed by general linear model for cooled semen and one-way ANOVA followed by LSD post-hoc test for post-thawed semen data using SPSS. For cooled semen, results showed that sperm motility and kinematics decreased (P < 0.05) in a time dependent manner, and the lowest value were recorded at 72 h. Supplementing semen extender with 20 and 30 µM lycopene improved total (48.0 ±% and 52.7±%, respectively) and progressive sperm motility (46.3±% and 51.6±%, respectively) than the control (44.9±8.5% and 42.3±7.8%, respectively) and other lycopene concentrations. Sperm kinematics tended to be higher in 20 and 30 µM lycopene than other concentrations. While for viability and membrane integrity were improved with 20 µM lycopene at 72 h post-cooling. Acrosome integrity showed the best results with 10 µM lycopene at 48 h. For post-thawed semen, 20 and 30 µM lycopene improved the total and progressive motility (34.83±1.32, 34.23±0.69, 31.61±1.13, 30.81±0.39, respectively) in comparison with 10 µM lycopene and control (25.85±2.79, 25.85±2.79, 22.63±2.47, 19.56±1.34, respectively. In conclusion, supplementation of semen extender with 20 or 30 µM lycopene improved sperm quality parameters in cooled and post-thawed ram spermatozoa.