The importance of polyunsaturated fatty acids (PUFAs) on chilled sperm quality is under investigation. Addition of fatty acids to the diet can influence sperm quality and male fertilizing capacity. The aim of this study was to investigate if the addition of n-3 PUFAs improves quality of bovine chilled sperm. In order to do experiment, 5 proven bulls were randomly selected and their ejaculates were collected by artificial vagina. Immediately, semen characteristics including volume, pH value, motility, viability, abnormality and concentration were recorded. Only ejaculates with normal characteristics were chosen. Then, each ejaculate was diluted by extender containing different supplementation (in 5 groups: group 1) basic extender (BX) as control, group 2) BX plus Polyethylene glycol (PEG) as a solvent, groups 3-5) BX containing three different concentration of n-3 PUFAs (1, 2.5 and 5%) in combination with PEG. Basic semen extender contains Tris-citrate buffer medium plus egg yolk and glycerol. All samples were maintained in refrigerator (5 ˚C) for 24 and 48 h. Motility (by CASA), viability and morphology were investigated in chilled sperm samples after 24 and 48 h. The results were evaluated by repeated measure ANOVA using SPSS and p<0.05 was considered significant. Immotility (D) increased and all other parameters decreased over the liquid preservation period within all groups including control. Although parameters were decreased during the next 24 hours of preservation, they were not significant. Different concentrations of omega-3 supplementation didn’t improve morphology and motility parameters significantly during the liquid preservation period. Even more, average of viability in group 5 (BX plus PEG and 5% PUFAs) was significantly decreased in compare with control group (P=0.001) which show an adverse effect on viability. Our results showed that PEG has some detrimental effects on sperm motility and viability during chilling, and the addition of n-3 PUFAs to semen extenders could not attenuate the detrimental effects of PEG and did not significantly improve bovine sperm quality. Nonetheless, diet supplementation of bulls with n-3 PUFAs is advisable to modify the fatty acid compositions of sperm plasma membrane for increasing its resistance to cooling

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