Functional and Flow Cytometric Analysis of Buffalo Cryopreserved Spermatozoa: Comparison of Different Breeds and Incubation Times

The purpose of this research was to compare the functional parameters of frozen-thawed Iranian Azaribuffalo spermatozoa with imported semen samples of Italian Mediterranean buffalo (IMB) after the thawing processand 4 hours of incubation. In this experimental study, a total of twenty-four ejaculates from four Iranian Azari buffalobulls were collected. Semen samples were diluted in AndroMed extender at a concentration of 50×106 spermatozoa/ml. The diluted samples were filled in 0.5 ml straws and were frozen in a programmable freezer. For imported semensamples, twenty-four samples of four IMB were used, which were diluted in AndroMed extender and frozen by the sameprocedure. Frozen-thawed sperm motion patterns, mitochondrial activity, membrane integrity, DNA integrity, reactiveoxygen species (ROS), and apoptosis status were evaluated immediately after thawing and 4 hours of incubation. Post-thawed sperm motility, progressive motility (PM), mitochondrial activity, membrane integrity weresignificantly higher in imported semen samples in compare with Iranian Azari buffalo. After 4 hours of incubation,sperm velocity patterns were superior in Iranian Azari semen samples. Moreover, the percentage of sperm cells withun-damaged DNA was higher in Iranian semen samples compared to imported samples at the time 0 of incubation.Following 4 hours of incubation, a significant increase in intracellular ROS level leads to reduced membrane integrity,mitochondrial activity, and DNA integrity in both buffalo breeds. At time 4, Iranian samples showed significantlylower apoptosis and higher dead spermatozoa compared to imported semen samples. Our study showed that the post-thawed quality of Iranian Azari buffalo semen was comparable with imported samples after 4 hours of incubation. Further investigations are recommended to assess the in vitro and in vivofertility rate of both buffalo breeds.