<div style="text-align:justify">Background: In order to find an alternative cryoprotectant for canine semen, different cryoprotectants, as dimethyl sulphoxide and ethylene glycol, dimethyle formamide either alone or in combination with glycerol have been evaluated on pooled semen of different breeds with different opinions. 
 
Methods: In the current study the effect of glycerol, DMF and their combination in Tris-citric acid-fructose egg yolk plasma extender on motility, viability, plasma membrane integrity (PMI), acrosome integrity (AI), inner mitochondrial membrane potential (IMMP), reactive oxygen species (ROS) and antioxidant enzymes in frozen-thaw semen of pug breed was compared. 
 
Result: Values for motility, viability, (PMI), AI were significantly (p&lt;0.05) high in TCFEYP-G compared to TCFEYP-DMF and TCFEYP-G+DMF at post thaw. However, HIMMP and MIMMP/MDA were non-significantly (P&gt;0.05) high and low in TCFEYP-G compared to TCFEYP-DMF and TCFEYP- G + DMF at post thaw, respectively. MDA concentration was significantly (p&lt;0.05) low in TCFEYP-G compared to TCFEYP-DMF and TCFEYP-G + TCFEYP-DMF extenders. However, H2O2, scavenging capacity of spermatozoa, cryopreserved in TCFEYP-G was significantly (p&lt;0.05) higher than in TCFEYP-DMF and TCFEYP-G + TCFEYP-DMF. However, there was no significant (p&gt;0.05) difference in superoxide free radical scavenging activity and nitrite concentration of spermatozoa among the three extenders. Activity of GPX, SOD and Catalase was also significantly (p&lt;0.05) higher in TCFEYP-G compared to TCFEYP-DMF and TCFEYP-G+DMF. However, values were reverse for GRE activity. This study concludes that glycerol is a healthier cryoprotectant to protect canine spermatozoa from cryo-injury during freezing-thawing process.</div>

Comparing Glycerol with Dimethylformamide and Combination for Cryopreservation of Pug Dog Semen in Egg Yolk Plasma Containing Extender