P-137: Effect of Different Equilibration, Freezing and Thawing Rate on Cryopreserved Buffalo Spermatozoa Diluted inTris- Egg Yolk Extender

Background: Artificial insemination (AI) has been used for increasing genetic potential of animals in production while application of AI has been restricted in buffalo due to low freezing ability and fertility of frozen-thawed buffalo spermatozoa. So improvement in buffalo sperm cryopreservation may enhance the breeding programs of this valuable farm animal. Semen processing is one of the major factors which affect post thawed sperm quality. Therefore the aim of this study was to evaluate sperm functional and structural characteristics in order to find optimal equilibration, freezing, and thawing rate of buffalo semen diluted in tris- egg yolk extender. Materials and Methods: Twenty ejaculates of four buffalo bulls were diluted in tris-egg yolk extender. Semen processing was done as follow: equilibration (2, 4, 8 and 16 hours), freezing (5, 10, 15 and 20 minutes), and thawing (30 seconds at 37°C, 15 seconds at 50°C and 6 seconds at 70°C). Post thawed motility characteristics, membrane integrity, Acrosome intactness and chromatin stability were assisted with CASA, HOS solution, Giemsa dye, and sperm chromatin structure assay respectively. Results: The post thaw sperm motility, plasma membrane integrity (PMI), and normal apical ridge (NAR) in equilibrium 4 and 8 hours were higher than the rest of the equilibration times. Ten minute freezing time significantly increased motility and progressive motility of spermatozoa compared to others freezing times (p<0.05). The motility and velocity patterns were superior in 70°C for 6 seconds as compared to 37°C and 50°C (p<0.05). DNA integrity of frozenthawed spermatozoa remained unaffected following different equilibration and freezing rates while sperm DNA damages were slightly increased when semen thawed in 70 °C for 6 seconds. Conclusion: The results showed that, 4 hours equilibration, 10 minute freezing time and 6 sec thawing in 70°C were suitable for buffalo sperm cryopreservation.