P-134: In Vitro Assessment of Cysteine Effect on DNA Integrity of Frozen-Thawed Buffalo Spermatozoa

Background: Cysteine has been used as additive in cryopreservation extenders. It contains of thiol group which plays an antioxidant role to eliminate reactive oxygen species (ROS) during freeze -thaw procedure. Excessive ROS production cause DNA damage and subsequently reduce sperm fertility. On the other hand, protamine protein have high levels of cysteine which is critical in packaging and compaction of sperm chromatin. To date there is no report regarding the cysteine effect on DNA integrity of buffalo spermatozoa following cryopreservation. Materials and Methods: Sixteen ejaculations of four mature buffalo bulls were spilt into four experimental groups and diluted with Tris - citrate -egg yolk extender which contains 0, 5, 7.5, 10 mM of cysteine. Frozen sperm were thawed and diluted with Tris -Null -EDTA buffer at the final sperm concentration of 5×106 cell/ml. 100 μl aliquots of this suspension was mixed with 200 μl detergent/acid solution. After 30 seconds acridine orange (AO) solution was added to the samples and cells were subjected to flowcytometry. The green fluorescence (intact DNA) detected by the FL1 detector was compared with red fluorescence (single strand DNA) detected by FL-3 detector ten thousand sperm cell were analyzed by cyflogic software. Results: Mean percentage of sperm with damaged DNA in cysteine 5, 7.5, 10 mM were 4.08 ± 0.69, 4.05 ± 0.85, 4.22 ± 0.18 respectively were reduced significantly in comparison with control group 6.2 ± 0.53 (p<0.05). Conclusion: It seems that 7.5 mM concentration of cysteine could improve DNA damage of frozen - thawed buffalo spermatozoa.