The aim of this study was to examine the effects of dietary supplementation with rumen protected n-6 or n-3 polyunsaturated fatty acids (PUFA) on the quantity and quality of semen from young post-pubertal dairy bulls. Pubertal Holstein-Friesian (n = 43) and Jersey (n = 7) bulls with a mean ± s.e.m. age and bodyweight of 420.1 ± 5.86 days and 382 ± 8.94 kg, respectively, were blocked on breed, weight, age and semen quality (based on the outcomes of two pre-trial ejaculates) and randomly assigned to one of three treatments: (i) a non-supplemented control (CTL, n = 15), (ii) rumen-protected safflower (SO, n = 15), (iii) rumen-protected n-3 PUFA-enriched fish oil (FO, n = 20). Bulls were fed their respective diets, ad libitum for 12 weeks; individual intakes were recorded using an electronic feeding system for the initial 6 weeks of the feeding period. Semen was collected via electro-ejaculation at weeks -2, -1, 0, 7, 10, 11 and 12 relative to the beginning of the trial period (week 0). On collection, semen volume, sperm concentration and progressive linear motility (PLM) were assessed. On weeks -2, -1, 0, 10, 11, 12, semen was packaged into 0.25 mL straws and frozen using a programmable freezer. On weeks -1, 7 and 11; a sub-sample of semen was separated into sperm and seminal plasma, by centrifugation and stored at - 20 °C until analysis of lipid composition. Semen from 10 bulls per treatment were used for post-thaw analysis at weeks 10, 11 and 12 (3 straws per ejaculate). Sperm motility was analysed by computer assisted semen analysis (CASA). In addition, membrane fluidity, acrosome reaction and oxidative stress were assessed using flow cytometry. Sperm from bulls fed SO had a 1.2 fold higher total n-6 PUFA content at week 11 compared to week -1 (P < 0.01) while bulls fed FO had a 1.3 fold higher total n-3 PUFA content, in sperm by week 11 (P < 0.01). There was no effect of diet on semen volume, concentration or PLM of sperm when assessed either immediately following collection or post-thawing. Membrane fluidity and oxidative stress of sperm were also not affected by diet. The percentage of sperm with intact-acrosomes was lower in CTL bulls compared to those fed SO (P < 0.01). In conclusion, while the lipid composition of semen was altered following dietary supplementation with either n-6 or n-3 based PUFA, this did not lead to measurable improvements in the quantity or quality of semen produced by young post-pubertal dairy bulls.