P-127: In Vitro Assessment of Trehalose for Cryopreservation of Ram Sperm in Extender Containing Soybean Lecithin

Background: Trehalose, a non-permeable disaccharide, has a protective role related both to osmotic effect and specific interactions with membrane phospholipids, rendering hypertonic media, causing cellular osmotic dehydration before freezing, and then decreasing the amount of cell injury by ice crystallization. Within this study, an attempt was made to evaluation of two levels of 50 and 100 mM trehalose in Tris based extender containing soybean lecithin on post-thaw quality of ram sperm. Materials and Methods: A Tris based extender containing 1% soybean lecithin and 7% glycerol was prepared and then divided to 3 aliquots. Then, different concentrations of trehalose (0, 50 and 100 mM) were added to extenders. Semen samples were collected by artificial vagina from four mature zandi native breed rams (3 and 5 yr of age) during the breeding season and after primary evaluation, samples were pooled in order to eliminate individual effect. The sperm were evaluated and accepted for evaluation if the following criteria were met: volume varying between 1 and 2 mL, sperm concentration of 3×109 sperm/mL, motile sperm percentage higher than 70%, and less than 10% abnormal sperm. The diluted semen was gradually cooled to for 2 hours. The cooled semen was loaded into 0.25-mL French straws (Biovet, L’Agile France). For assessment of sperm after freezing, the sperm were thawed and then prepared for evaluation. Motion parameters were assessed by CASA. Viability and membrane integrity and abnormality were evaluated by eosin-nigrosin, Host and Hanckok solution, respectively. Results: The extender supplemented with 50 mM trehalose exhibited the greatest percentages of sperm motility (58.43 ± 1.86), membrane integrity (52.34 ± 1.46), viability (63.71 ± 1.52), in compare to 0 (48.71 ± 1.86, 45.46 ± 1.46, 56.71 ± 1.52, respectively) and 100 (44.57 ± 1.86, 41.01 ± 1.46, 55.29 ± 1.52 respectively) mM trehalose (p<0.05). Also, 50 mM trehalose significantly reduced percentages of post-thaw total abnormalities (14.94 ± 1.16) compare to control (19.25 ± 1.16) and 100 mM trehalose (20.13 ± 1.16). Conclusion: The findings of this experiment show that when the trehalose in the diluents containing lecithin 1% and glycerol 7% a concentration of 50 mM had better performance than to 100 mM, and significant improve sperm mobility parameters.