This study analyzed the effects of dietary sources of omega-3 and omega-6 fatty acids on semen parameters and fertility potential in broiler breeder roosters. The mRNA and protein profiles of peroxisome proliferator-activated receptors-γ (PPAR-γ) expression in sperm as potential mediator of FAs were considered. Roosters were categorized into three groups and received their diets for 24 weeks as follows: 1) control diet received a basal diet (CTRL); 2) Fish oil based diet (FO) received the basal diet supplemented with 15 g/kg of diet fish oil; and 3) sunflower oil based diet (SO) received the basal diet supplemented with 15 g/kg of diet sunflower oil. While the different diets had significant effects on semen parameters, the effect of sampling time was not significant. The effect of the diets on sperm parameters were significantly higher in the SO and FO groups in total motility, progressive motility, amplitude of lateral head displacement, linearity, straightness, wobble and viability (P ≤ 0.05). Fertility rate was significantly improved in the FO and SO groups (P = 0001). The highest value for PPAR-γ mRNA was observed in the SO group compared to other groups (P ≤ 0.05). Moreover, supplementation of the roosters’ diets with FO and SO increased PPAR-γ protein expression compared to the control. It seems that PPAR-γ could be a strong potential mediator of the underlying mechanism of improvement in semen parameters and reproductive performance of roosters under the effects of both dietary omega-3 and omega-6 polyunsaturated fatty acids.Copyright © 2020 Elsevier Inc. All rights reserved.