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 30ËšC 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.