The objective of this study was to examine the effect of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation of rams on semen quality and subsequent sperm function of liquid stored semen. Mature rams of proven fertility were individually housed and were blocked according to breed, body weight, and body condition score and randomly allocated within block to one of two dietary treatments (N = 7 per treatment). Rams were offered a base diet of hay and concentrate, with the concentrate enriched with either: (1) saturated palmitic acid (CON) or (2) high n-3 PUFA fish oil (FO) supplements. Both lipid supplements were added at 2% (wt/wt) of the total diet as fed and both were partially rumen-protected. The animals were fed their respective diets for a total of 9 weeks and blood samples were collected on weeks 0 (pre-experimental), 4, and 9, relative to initial allocation of diet (week 0), for measurement of plasma concentration of fatty acids, metabolites, insulin like growth factor 1 (IGF-1) and insulin. Semen was collected from each ram (on 1 day in each week) in weeks 4, 5, 7, 8, and 9, and each ejaculate was assessed for volume, wave motion, and concentration of sperm, after which it was diluted in a skim milk-based extender and stored at 4 C. A second ejaculate was collected on weeks 4, 7, and 9, centrifuged, and the sperm frozen for subsequent lipid analysis. A sample of semen from each ram was assessed at 24, 48, and 72 hours after collection for sperm progressive linear motion, ability to penetrate artificial mucus, and the ability to resist lipid peroxidation (at 24 and 48 hours only) using the thiobarbituric acid reactive substances assay. There was no effect of diet on plasma insulin concentrations or on any of the metabolites measured, however, there was a diet by week interaction for plasma IGF-1 concentration (P < 0.05). This was manifested as the FO supplemented rams having higher IGF-1 concentrations on week 9 compared with the control treatment (P < 0.05), but not at the earlier sampling dates. Compared with the pre-experimental values, supplementation with FO increased plasma concentrations of total n-3 PUFAs by 3.1-fold and decreased n-6 PUFA concentrations by 1.84-fold. Consequently, the ratio of n-6 to n-3 PUFA was decreased in the FO-supplemented rams (P < 0.001). Dietary supplementation with FO increased the concentration of eicosapentaenoic acid in sperm from week 4 to 9 by 2.7-fold (P < 0.05) leading to a 1.5-fold increase in total n-3 PUFA in the same period. Ejaculates collected from rams supplemented with FO yielded a higher semen concentration (P < 0.05), however, there was no difference between diets on any of the other semen quality parameters including semen volume, wave motion, progressive linear motion, ability to penetrate artificial mucus, or ability to resist lipid peroxidation. In conclusion, dietary supplementation of rams with n-3 PUFA successfully increased the n-3 PUPA content of plasma and sperm but has limited effects on the quality of liquid stored semen. (C) 2014 Elsevier Inc. All rights reserved.