**3.2.2 Acrosomal membrane integrity**

Acrosome is the acidic secretory organelle filled with hydrolytic enzymes. Assessment of the acrosomal status is a very important part of semen evaluation, in the view of the role of this structure in the maintenance of spermatozoal ability to penetrate the egg's zona pellucida (in mammals), or the egg envelope (in birds) and the ability to fuse with the egg plasma membrane. Cells must retain a normal acrosome to ensure that the acrosome reaction may occur at the suitable time to facilitate fertilization (Esteves et al., 2007). Also the determination of the acrosome status in cryopreserved sperm is of the fundamental importance as cryopreservation directly damages sperm membrane, which could be followed by a loss of the acrosomal matrix contents.

Acrosomal status may be assessed using lectins, such as peanut agglutinin from *Arachis hypogaea* (PNA) or *Pisum Sativum* agglutinin (PSA), conjugated with different fluorescent probes like fluorescein isothiocyanate (FITC), phycoerythryn (PE) or Alexa Fluor®, (Graham et al., 1990; Kawakami et al., 2002; Nagy et al., 2004; Partyka et al., 2010; Peña et al., 2001; Rijsselaere et al., 2005). For human sperm concanavalin A lectins (ConA) is used as well (Holden et al., 1990). The PNA labelling is specific for the outer acrosomal membrane and it binds to β-galactose moieties. Whereas the PSA is labelling α-mannose and α-galactose moieties of the acrosomal matrix. The absence of the fluorescence on the living sperm is indicative for an intact acrosome, and fluorescence is indicative for acrosome disruption or acrosome reaction (Silva & Gadella, 2006). Since PNA agglutinin displays less non-specific binding to other areas of the spermatozoa, it leads some researchers to favour this over PSA (Graham, 2001). Lectins may be also combined with Hoechst 33258, carboxy-SNARF/PI, ethidium homodimer allowing for simultaneous assessment of acrosomal status and membrane integrity (Fig. 6b) (Kawakami et al., 1993; Szász et al., 2000).
