**7.4 Acrosome integrity**

Acrosome integrity will be assessed using fluorescein isothiocyanate–labeled peanut (*Arachis hypogaea*) agglutinin (FITC-PNA) staining. Ten µl of the diluted semen is mixed with 10 µl of Ethidiumhomodimer-1 and incubated at 37 ºC for 15 min. Five µl of the mixture is smeared on a glass slide and fixed with 95 % ethanol for 30 second. Fifty µl Fit C-PNA (dilute Fit C-PNA with PBS 1:10 v/v) is spread over the slide and incubated in a moist chamber at 4 ºC for 30 min. After incubation, it is rinsed with cold PBS and air dried. Two hundred spermatozoa are assessed under fluorescent microscope at 1000x magnification and classified as intact acrosome, damaged acrosome and missing acrosome (Cheng et al., 1996; Axner et al., 2004). The results are scored as the percentage of intact acrosome spermatozoa.
