The successful of artificial insemination (AI) in buffalo is still low. The quality of frozen semen
determines the successful of AI program. Glycerol is widely used for cryopreservation of buffalo semen with unsatisfactory results due to its toxicity and antifertility properties. This study aimed to investigate the effect of glycerol and dimethylformamide (DMF) concentrations in skim milk egg yolk
(SMEY)–based diluent on the quality of frozen-thawed swamp buffalo bull semen. Fresh semen was
divided into eight aliquots. Each aliquot was diluted in SMEY containing 4%, 5%, 6%, and 7% glycerol and 4%, 5%, 6%, and 7% DMF. Diluted semen was packed into mini straw (0.25 mL), equilibrated
at 4 °C for 4 hours, frozen in liquid nitrogen vapor for 10 minutes, and stored at liquid nitrogen
container. The straws were thawed after 24 hours at 37 °C for 30 seconds for evaluation. The results
showed that the post thawed sperm motility and recovery rate in SMEY extender containing 7% glycerol and 5% DMF were higher than the other glycerol or DMF concentrations. Post thawed sperm viability and plasma membrane integrity in SMEY containing 4%-6% DMF did not differ significantly,
however both values were higher than those in 7% DMF. Post thawed sperm viability and plasma
membrane integrity did not differ between SMEY containing 6% and 7% glycerol. However, both
values were higher than those in 4% and 5% glycerol. It is concluded that 7% glycerol or 5% DMF is
considered to be the most suitable cryoprotective agent for swamp buffalo semen cryopreservation.