Cryopreservation is the freezing of cells or tissues to subzero temperatures, typically -196 º C. Many benefits have resulted from the process of cryopreservation. Damage induced by cryopreservation has been results cold shock, oxidative stress, osmotic changes, and formation of ice crystal and lipid–protein reorganizations within the cell membrane. Oxidative damage caused by reactive oxygen species (ROS) leads to impaired cell functions. Free radicals, includes ROS and RNS, are normal pro - oxidant molecules in aerobic metabolism. Alpha lipoic acid is a non-vitamin coenzyme that helps in significant metabolic and antioxidant functions in the body. Alpha lipoic acid has been reported to have extra functions by which they are able to synthesize vitamin C from its reduced form in the presence of glutathione. It is matchless among biological antioxidants, because it is equally lipid and water soluble. This allows it to nullify free radicals almost everywhere in the body, inside as well as outside the cells. Therefore, the objective of present study is to determine the effect alpha lipoic acid on post thaw quality and in vitro incubation of buffalo bull semen. Alpha lipoic acid scavenge on reactive oxygen species formed in semen during the process of cryopreservation, so it maintained good semen quality during post thaw and in vitro incubation. Three mature Nili-Ravi buffalo (Bubalis bubalis) bulls (4-8 year age) kept at SPU, Qadirabad Sahiwal Pakistan were used in the study. These bulls are being used as regular donors at SPU. There semen was collected with artificial vagina of temperature 42c; three ejaculates (one from each) was pooled and diluted (30 million sperms/ml) with extender of different inclusion levels (0.0, 0.5, 1, 1.5, 2, 2.5 mmol/ml) of alpha lipoic acid. Straws were filled and extended then semen was cooled for 2 hours and equilibrated for two hours. Semen was placed in Liquid nitrogen vapors for 10 minutes. Finally semen straws was put in liquid nitrogen, Total five replicates were performed. Now post thaw quality was checked in
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which various tests were performed, like %age motility, Acridine orange assay for DNA integrity, HOST for plasma membrane integrity, Fitc-PNA/PI for viability and acrosomal integrity. Longevity test was performed by in vitro incubation of frozen thawed semen sample in SOF and evaluating it at 1.5, 3 and 4.5 hour interval in Carbon dioxide incubator. It was expected that Alpha lipoic acid shown positive effect on post thaw quality and in vitro incubation of buffalo bull semen, in the meaning of increased percentage motility, Less DNA damage during cryopreservation and incubation, Increased acrosomal and plasma membrane integrity. So alpha lipoic acid shown positive effect by counter acting on ROS during cryopreservation and in vitro incubation. Results acquired from this study shown that an increase in sperm motility, plasma membrane integrity, DNA integrity, Acrosomal integrity, viability and survival was caused by ALA competences in energy production and anti-oxidant properties, when used at the concentration of 0.5mM and 1mM. In summary, based on the results of our study, it can be concluded that an optimal concentration (0.5mM and 1mM) of ALA improved PMI, sperm motility and viability, minimize DNA damage and improved sperm survival.