The aim of this study was to investigate the effects of several dietary fatty acids (FAs) on semen quality and blood parameters in rams. We gave diet-supplemented treatments (35 g day−1 ram−1) by C16:0 (palm oil), C18:2 [sunflower oil (SO)] and an n-3 source [fish oil (FO)] to 12 rams, who were fed for 15 weeks during their breeding season. Semen was collected once per week. Semen samples were extended with Tris-based cryoprotective diluents, then cooled to 5 °C and stored in liquid nitrogen. Positive responses were seen with FO after 4 weeks. The mean prefreezing semen characteristics improved with the intake of FO (P < 0.05). Interestingly, maximum sperm output in FO was achieved 7.5 × 109 when compared to palm oil 5.3 × 109. Rams that received FO had the highest total testosterone concentrations (11.3 ng ml−1 for FO, 10.8 ng ml−1 for SO and 10.2 ng ml−1 for palm oil) during the experiment (P < 0.05). FO also improved the rams' sperm characteristics after thawing (P < 0.05). Although C16:0 is a major saturated FA in ram sperm and all rams have been fed isoenergetic rations, the unique FAs of FO improved fresh semen quality and freezing ability compared to other oils.