This study aimed to assess the sperm morphology in 198 bull ejaculates, comparing the results obtained in smears stained with eosin-nigrosin (EN) evaluated under bright field microscopy and in wet semen smears fixed with buffered formol-saline and observed under phase contrast microscopy (FS). In addition, the suitability of EN for assessment of shape and size morphology of the sperm head was also evaluated by comparing this method against results obtained in samples stained with carbol-fucsin (William’s staining) (CF). Sperm morphology was determined in all methods assessed by the same operator through counting 400 spermatozoa per sample.

EN detected a lower percentage of total acrosome abnormalities compared with FS (2.90±3.2% vs. 8.45±6.8%, P< 0.0001). Specifically, the difference was observed for damaged and knobbed acrosomes (1.34±1.9% vs. 5.33±5.0%, P< 0.0001 and 1.69±2.5% vs. 3.20±4.7%, P= 0.0001 respectively).

Although the total % of head shape and size abnormalities determined by EN and FS was not statistically different (3.16±8.4% and 2.25±6.3% respectively, P> 0.05), there was a significant difference for the total percentage of nuclear defects between methods (0.69±1.1% vs. 4.47±8.8%, P< 0.01). In detail, this difference was for the crater defect (0.25±0.8% vs. 0.92±2.2%, P< 0.0001), single nuclear pouches (0.04±0.2% vs. 1.60±4.3%, P< 0.0001) and diadem (0.12±0.2% vs. 1.88±5.9%, P< 0.0001).

Lower percentages of abnormal mid-pieces (0.05±0.2% vs. 0.12±0.2%, P< 0.05) and distal droplets (1.99±4.0% vs. 3.21±5.3%, P< 0.05) were also observed with EN compared to FS. For the remaining sperm defects evaluated, no significant differences were found using either technique (P> 0.05). Likewise, the percentage of head shape and size defects when comparing EN with CF was not statistically different (3.16±8.4% and 2.91±8.4% respectively, P> 0.05).

EN is currently widely used for evaluation of sperm morphology under field conditions since it is a method of simple preparation and given that it needs an undemanding equipment (bright field microscopy). However, the underestimation when using this method of uncompensable abnormalities such as nuclear craters (3.6 times less), single nuclear pouches (40 times less) and diadems (15.7 times less), compared to FS, emphasizes the later as the method of choice when evaluating sperm morphology under field conditions in bulls extensively reared.