This study was carried out at El-Gemmezah Animal Production Research Station, belonging to Animal Production Research Institute, during the period from November 2006 to January 2007 to investigate the effect of adding different types and levels of cryoprotectants on sperm viability during different freezing processes of buffalo semen. Five sexually mature buffalo bulls aged 7-10 years were used for semen collection by means of an artificial vagina. Ejaculates were obtained from each buffalo bull twice/week for 10 collection weeks (100 ejaculates). The main extender used for semen dilution was Egg Yolk-Citrate-Tris with different types (glycerol, GL; dimethyl sulfoxide, DMSO and ethylene glycol, EG) and levels (5, 7 and 10% for each) of cryoprotectants. Then, semen was frozen in liquid nitrogen and thawed at 37°C/30 sec. Percentage of progressive motility of spermatozoa was determined pre- and post-dilution, post- equilibration period for 4 or 6 h, post-thawing after 24 h freezing period. Results showed that in post-diluted semen, sperm motility was the highest (72.9%, P<0.05) with GL, followed by DMSO (98.8%), while, EG showed the lowest motility (63.6%). The differences between DMSO and each of GL and EG were not significant. Increasing level of cryoprotectant resulted in gradual reduction in sperm motility, being significant (P<0.05) only by increasing the level from 7 to 10%, whereas sperm motility slightly decreased from 70.7% at a level of 5% to 69.3% at a level of 7%, while it decreased (P<0.05) to 65.2% at a level of 10%. The effect of interaction between type and level of cryoprotectants on sperm motility in post-diluted semen was not significant. In post-equilibrated semen, sperm motility with GL or DMSO was higher (68.7 and 64.8%, respectively, P<0.05) than that with EG (557%). Sperm motility with GL or DMSO did not differ significantly. Increasing level of cryoprotectant from 5 to 7% resulted in slight and insignificant reduction in sperm motility from 65.1 to 64.9%. This reduction was significant (P<0.05) by increasing the level from 7 to 10%, being 59.2% at a level of 10%. Sperm motility was higher (P<0.05) with 6 than 4 hours (58.9 vs. 67.2%) as equilibration period. Only the effect of interaction between type and level of cryoprotectants was significant (P<0.001) on sperm motility in post-equilibrated semen. In post-thawed semen, sperm motility was the highest (36.4%, P<0.05) with GL, moderate (29.7%) with DMSO and the lowest (9.5%) with EG. Increasing level of cryoprotectant from 5 to 7% resulted in slight and insignificant increase in sperm motility in post-thawed semen from 27.4 to 28.8%. Sperm motility sharply decreased (P<0.05) by increasing the level from 7 to 10%, being 17.6% at a level of 10%. The effect of interaction of type with level of cryoprotectants was not significant on sperm motility in post-thawed semen.
The current study concluded that glycerol was the best cryoprotectant as compared to dimethyl sulfoxide and ethylene glycol when it was added at a level of 7% in Tris-based extender used for freezing Egyptian buffalo bull semen.