Effects of Extender Osmolarity, Cooling Rate, Dilution Rate and Glycerol Addition Time on Post-thaw Ram Semen Characteristics and Fertilization

Tree experiments were designed to optimize ram semen cryopreservation and to examine the effect of the extender b osmolarity, extension time with extender a and b, presence of glycerol in freezing media, cooling rate from 30°c to 5°c, and dilution rate on post-thaw ram semen characteristics and fertilization. ram ejaculates of thick consistency with rapid wave motion (>+++) and >70% initial motility were pooled. pooled semen was diluted with tris based extender using a two-step dilution method. semen motility and morphology related parameters were assessed at the following five steps : (1) fresh, (2) after dilution with extender a, (3) at 5°c, (4) after equilibration, and (5) after thawing at 37°c for 30 min. the extender osmolarity affected significantly post-thaw semen motility, defected acrosomes (da) and total morphological defect (tmd), generally. there was a significant interaction between the osmolarity, cooling rates, glycerol addition time and first dilution time, on post-thaw motility, da and tmd. higher fertilization rates were obtained in fresh semen and group 15 compared to group 21 (p<0.05). in conclusion, cooling rate, extender osmolarity, presence of glycerol, dilution time with extender a and b, and dilution rates interact on post-thaw semen parameters. glycerol included freezing media improves post-thaw semen recovery. also, increasing the extender osmolarity resulted better post-thaw semen motility especially in the group frozen without glycerol. post-thaw semen parameters not affected by cooling and glycerol addition time regimes, generally. obtained data showed that presence of glycerol in freezing media have a negative effect on embryonic development and it was observed that frozen ram semen at high osmolarity without glycerol could be used for in vitro fertilization.