This study was aimed to investigate the effects of omega-3, 6, 9 fatty acids on the characteristics of bovine chilled and frozen-thawed semen. For this purpose, oil containing different levels of omega-3, 6, 9 fatty acids were added to semen extender. To emulsify the oil in semen extender, polyethylene glycol (PEG) was added as a suitable solvent and the solution was finally sonicated. Five proven Holstein bulls were randomly selected and their ejaculates were collected using an artificial vagina. Groups were designed as control, treatments 1, 2, 3 and 4. The control group contained only the basic extender (Tris-citrate buffer, egg yolk and glycerol) without any additives. In treatment 1, only 5% PEG was added to the diluent; while in treatments 2, 3 and 4 different concentrations of omega-3, 6, 9 fatty acids (1.0, 2.5 and 5.0%) in combination with PEG were added to the basic extender. After dilution, the semen samples were packaged into 0.5 ml straws, a process that was followed by cooling the semen straws. Motility, viability and morphology of semen samples were evaluated after 24 and 48 h of storage in refrigerator (5 ˚C) or after one month of storage in the liquid nitrogen. Immotility was increased and all the other parameters including motility, viability and morphology were significantly decreased in all the groups compared with fresh samples during cold storage and freezing-thawing. Our results demonstrated the following: 1) PEG has significant detrimental effects, especially on the sperm motility; 2) addition of omega-3, 6, 9 fatty acids could not improve the sperm motility in chilled storage condition and after freezing-thawing; and 3) omega-3, 6, 9 fatty acidscould not also attenuate the other deleterious effects of PEG. In conclusion, our findings reveal that addition of these fatty acids to the semen extender does not enhance the resistance of the bovine sperm membrane to cooling and freezing-thawing and that further studies are required to find suitable candidate compounds that can boost the quality of semen that is chilled and freeze-thawed.

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