In many studies are being conducted to improve fertility of bull sperm. The increasing of sperm membrane unsaturated fatty acids can enhance sperm quality. The aim of this study was investigation about effects of omega- 3,6,9 fatty acids on characteristics bovine chilled sperm. For this purpose several levels the oil containing omega- 3,6,9 fatty acids was added to semen extender. In this experiment 5 proven bulls were randomly selected and their ejaculates were collected by artificial vagina. Semen was analyzed in terms of several parameters including volume, concentration, motility, viability and normal morphology immediately. To emulsify the oil in semen extender, polyethylene glycol (PEG) was added as a more suitable solvent and solution was finally sonicated. Experimental groups included: control and treatments 1, 2, 3 and 4 as sham, levels 1, 2.5 and 5% of omega-3,6,9 fatty acids, respectively. In treatment 1, PEG was added alone to the diluents and in treatments 2, 3 and 4 different concentration of omega-3,6,9 fatty acids in combination with PEG were added to extender based Tris- citrate buffer, egg yolk and glycerol. After dilution, semen samples were packed into 0.5 ml straws and then cooling process was performed. Samples were measured in terms of Motility, viability and morphology after 24 and 48 h that hold in refrigerator. The results were evaluated by repeated measure ANOVA and p<0.05 was considered significant. Motility and other dynamic parameters were analyzed by computer aided sperm analyzer (CASA) and viability and morphology were measured using phase contrast microscope. Motility, Viability and normal morphology were 47.19, 27.97, 30.86, 26.02 and 23.41% and 86, 77.75, 81.2, 73.8 and 60.75 % and 91.8, 90.2, 90.75, 90 and 89.75% after 24h and 38.43, 21.98, 19.53, 15.18 and 14.69% and 80.2, 64, 63.6, 60.6 and 61.5% and 90.4, 87.8, 88.8, 86 and 89% after 48h for control, treatment 1, 2, 3 and 4 respectively. Our result showed that PEG has detrimental effects on motility significantly (p<0.05) while this effects was not significant on the viability and morphology(p> 0.05). Moreover, we found that combination of omega-3,6,9 fatty acids cannot improve sperm motility (p<0.05), viability (p<0.05 for treatment 4 only) and morphology (p> 0.05) after 24 and 48 h. In conclusion, this fatty acid compound could not improve sperm charactristics in cold condition. In addition, using lower percentage of PEG as well as replacement of a part of glycerol by PEG and decreasing levels of fatty acids might give better results.

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