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.