This research aimed to evaluate the effect of inclusion unsaturated fatty acids (UFAs) in the buffalo diet, on the sperm quality after thawing. In parallel, it was verified the degree of association among techniques to assess plasma membrane integrity (PMI) of spermatozoa. Patterns of sperm motility (subjective and computer-assisted analysis), kinetics, morphology and plasma membrane integrity were evaluated. The hypo-osmotic swelling test, nigrosin-eosin staining (E-N) and fluorescent probes propidium iodide/Hoechst 33342 (IP-H342) were adopted to evaluate PMI. The study was planned considering two experimental groups, which were evaluated in repeated measures design. over time. Eleven Murrah buffalo bulls (3.5±1.0 years, and 536.9±73.0 kg) were divided in Control Group (CONT, n=5) and Palm Oil Group (PO, n=6). Animals were fed with forage and concentrated in a controlled manner. Animals of PO received dietary supplementation containing palm oil (2% of dry matter). Dietary consumption was daily monitored and the intake level of UFAs in PO was 55.4% higher than CONT. Semen samples were collected and evaluated every 14 days. The experiment lasted 120 days, considering four periods of 30 days each (Periods I, II, III and IV) and repeated measures structure was used in statistical analysis. Significance level adopted was 5%. There was no effect of treatment on sperm motility (P>0.05), but palm oil supplementation presented a positive effect on spermatic ability to progress (derived from BCF, STR and LIN) in Period IV (P<0.05). High correlation (91%) was observed between IP-H342 and E-N techniques (Y = 0.9155 x + 2.9843, R2 = 0.82, P?0.05). Thus, supplementation with UFAs increased the progressivity of spermatozoa after 90 days, and had no effect on other seminal parameters. Eosin-nigrosin stain can be used efficiently as a predictor of the level of PMI when conditions for implementing fluorescent probes not present.