Improvement of post-thaw quality of Donkey (Equus asinus) semen is essential to augment the in-vivo and in-vitro fertilization rate and to be used for mule production. By the help of cryopreservation sperm cells can be stored for the long time but it causes lethal sub-lethal damage to the sperm. In most species including Donkey and horses sperm cryosurvival rates are not optimal because of its plasma membrane composition. One of the major cryopreservation damage is produced by Reactive oxygen species (ROS) generating oxidative stress caused by ROS are important for normal sperm function but in normal concentration. When they are produced in more quantity they cause damage to Acrosome, DNA and plasma membrane. . 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 was to determine the effect alpha lipoic acid on post thaw quality of jack 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. Two adult donkeys (Equus asinus) (4-6 years old) kept at animal shed Ravi campus pattoki were used in the study. All the animals were managed under optimal condition of feeding and management. Donkeys were offered green fodder with ad libitum supply of water. Semen collection was done twice a week (one ejaculate/collection) using an equine artificial vagina having temperature of 45-50 ºC. Five collection from each donkey were done (n=10). Ejaculates were filtered with muslin cloth to remove gel. Semen volume was measured by collecting semen in a graduated collection tube after
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filtration and the sperm concentration was measured by using a phase contrast microscope (40 x, Nikon) and was scored with a coverslip and then immediately was kept in water bath having 37 ºC temperature after collection until evaluation and processing. Semen quality parameters like volume, concentration and motility were recorded. After initial evaluation, semen samples were extended with centrifugation extender in 1:1 and seminal plasma was removed after centrifugation. Supernatant was removed so that seminal plasma up to 20% will remain with sperm pellet and was maintained at 37 ºC temperature in water bath and was extended with extender having different concentrations of Alpha lipoic acid (0mM, 0.5mM, 1mM, 1.5mM, and 2mM) and cooled for 2 hours and then equilibrated for 2 hrs at 4oC. Then, French semen straws of 0.5ml capacity were filled with semen (100x106/straw). All semen straws were arranged on a rack and then placed at 4cm height above liquid nitrogen vapors for 10 minutes. Finally semen straws was put in liquid nitrogen, Total five replicates per donkey were performed. Now post thaw quality was checked in 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. It was expected that Alpha lipoic acid shown positive effect on post thaw quality of donkey 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 by reducing melondialdehyde production as indicated by MDA test carried out in this study. 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 1.5mM. In summary, based on the results
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of our study, it can be concluded that an optimal concentration (1.5mM) of ALA improved PMI, sperm motility and viability, minimize DNA damage and improved sperm survival.