Nerve growth factor-β (NGF) is a seminal plasma protein associated with improved sperm membrane integrity and motility in mammalian species. Greater NGF concentration has been identified in the seminal plasma from bulls with positive sire conception rate. Seminal plasma NGF mRNA expression was also found to be positively associated with the maintenance of post-thaw functional membrane integrity in bull spermatozoa, suggesting that it could impact cryotolerance. Semen collected by electroejaculation (EEJ) and epididymal-harvested sperm (EPI) are not the most common methods used in the AI industry, but both methods can be models to test the NGF impact on cryotolerance. Our objectives were to compare post-thaw semen quality from EEJ and EPI from bulls with purified NGF before cryopreservation. Semen was obtained from Angus x Simmental crossbred bulls (n = 10) by EEJ and EPI 3 d later. Semen samples were incubated with a commercial extender having 0 ng/mL (CONT), 0.5 ng/mL (LOW), 5 ng/mL (MED), or 50 ng/mL (HIGH) of purified NGF before cryopreservation. Sperm motility was assessed in each sample before treatment and cryopreservation and at post-thaw. Flow cytometry was used for post-thaw assessment of sperm viability, acrosome integrity, and chromatin stability. Kruskal-Wallis rank sum test and ANOVA were used for the statistical analysis. Post-thaw sperm motility and velocity parameters (VCL) were decreased, while linearity (LIN) was increased in HIGH versus CONT EEJ samples (P < 0.01), but no differences were observed in EPI samples (P = 0.22). HIGH EEJ samples had a lower amplitude of lateral head displacement (ALH) at 2.5 and 3 h post-thaw (P < 0.01). Post-thaw sperm viability, acrosome integrity, and DNA fragmentation index were not affected by NGF treatment in either EEJ or EPI (P ≥ 0.15). Treatment with NGF did not improve cryotolerance of sperm collected by electroejaculation or epididymal harvest in bulls. Supplementation with high concentrations of NGF decreased post-thaw VCL and ALH and increased LIN, which may suggest a role in preventing premature sperm hyperactivation and capacitation.