**3.2.4 Capacitation status**

Before fertilizing of the oocyte, mammalian spermatozoa undergo the sequence of membrane alterations associated with accumulation of calcium ion and the increase of tyrosine phosphorylation resulting in sperm hyperactivation (Hewitt & England, 1998; Petrunkina et al., 2003). At the contact with oocyte, capacitated spermatozoa presents the acrosome reaction which enables the zona pellucida penetration. However, in avian spermatozoa it is believed that a period of capacitation within the female's reproductive tract in order to fertilize ova is not required (Howarth, 1971). The hen oocyte is not surrounded by cumulus cells that would require a different way of sperm motility to pass them trough. It may therefore be suggested that there is no need for motility hyperactivation to prepare for the acrosome reaction in the chicken and that this special motility pattern has not been developed in birds (Lemoine et al., 2008).

The capacitation of the mammalian spermatozoa is assessed by using chlorotetracycline assay (CTC), lectins, measurements of CASA motility characteristics and assessment of thyrosine phosphorylation within plasma membrane (Guérin et al., 1999; Hewitt & England,

Methods of Assessment of Cryopreserved Semen 559

Effect of oxidative stress is particularly important during the storage of sperm and its cryopreservation. The analysis of semen of mammalian and avian species, showed that the production of ROS and LPO occurrence is increased during freezing- thawing (Bilodeau et al., 2000; Chatterjee and Gagnon, 2001, Guthrie & Welch, 2007; Neild et al., 2005; Partyka et al., 2011b). The main site of their formation are mitochondria (Brouwers & Gadella, 2003) and sperm cell membranes (Agarwal et al., 2005), which are particularly vulnerable to damage from sudden temperature changes. Although, aerobic cells have substrates and enzymes to prevent or restrict the formation and propagation of ROS, but the antioxidant defence of spermatozoa are relatively weak and these germ cells are very susceptible to

As an alternative to the colorimetric detection of lipid peroxide formation, a fluorescent membrane probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3 undecanoic acid (C11-BODIPY581/591) has recently been successfully used in human, equine, bovine, porcine, feline and chicken's and goose's spermatozoa (Aitken et al., 2007; Almeida & Ball, 2005; Brouwers & Gadella, 2003; Brouwers et al., 2005; Neild et al., 2005; Partyka et al., 2011a,b; Thuwanut et al., 2009). This is an oxidation-sensitive fluorescent fatty acid analogue, that is easily incorporating into membranes and fluoresces red in the intact state, but turns green after undergoing peroxidation (Drummen et al., 2002). C11-BODIPY581/591 oxidation is virtually insensitive to environmental changes and the probe does not spontaneously leave the lipid bilayer after oxidation, moreover the extent of peroxidation is correlated with the formation of hydroxyl- and hydroperoxiphosphatidylcholine (Brouwers & Gadella, 2003; Brouwers et al., 2005). The degree of probe peroxidation can be followed in separate sperm subpopulations using flow cytometry, or localized in individual sperm using fluorescence microscopy. Moreover, the use of combination C11-BODIPY581/591 with PI makes it possible to distinguish the presence of reactive oxygen and nitrogen species in the

hydrophobic part of lipid bilayers of live sperm from dead cell membranes (Fig. 6d).

For monitoring the intracellular level of ROS, such as hydrogen peroxide (H2O2) in the spermatozoa, the fluorescent dye 5-(and-6)-carboxy-20,70-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) can be used. Viable spermatozoa are differentiated from dead cells by a counterstain - propidium iodide and the subpopulations of sperm with a high H2O2 level (strong fluorescence) and with low H2O2 level (weak fluorescence) can be

Apoptosis is a physiological mechanism required for any organism function. In contrast to necrosis, apoptosis is a process, where cells play an active role in their own death. Apoptosis comprising of a complex phenomenon that includes three stages: induction, execution and degradation. The most significant changes related to apoptosis are the externalization of the phosphatidylserine (PS), DNA fragmentation, caspase activation, loss of mitochondrial membrane potential, and increase in sperm membrane permeability (Bratton et al., 1997; Glander & Schaller, 1999; Martin et al., 2004;). Several pathways are reported for mammalian cell apoptosis. These include the intrinsic, extrinsic, and apoptosis-inducing factors. During the early phases of disturbed membrane function, asymmetry of the membrane phospholipids occurs, before the integrity of the plasma membrane is

oxidative stress (Jones & Mann, 1977).

distinguished.

**3.2.6 Apoptotic changes** 

1998; Petrunkina et al., 2004; Rota et al., 1999). Fluorescent antibiotic CTC is used to assess the destabilization of sperm membrane. Neutral and uncomplexed CTC crosses over the cell membrane, enters intracellular compartments and binds to free calcium ions. During these events, CTC becomes negatively charged and after creating CTC-Ca+2 complexes becoms more fluorescent. Thus CTC can be used as a tool to distinguish capacitated and uncapacitated spermatozoa. Three classes of sperm cells may be assessed: uncapacitated and acrosome intact (F-pattern, an overall staining of the sperm head), capacitated and acrosome intact (B-pattern, a prominent staining of the apical area of the sperm head) and capacitated and acrosome reacted (AR-pattern, loss of staining of the sperm head) (Maxwell & Johnson, 1997). CTC may be combined with Hoechst 33258, to simultaneous assessment of percentage of live cells and capacitation status (Hewitt & England, 1998).

The exposure of spermatozoa to low temperatures shortens their capacitation time, changing the membrane lipid architecture, membrane permeability and the reducing efficiency of enzymes extruding calcium ions. These changes resemble capacitation, and are likely to reduce long-term sperm viability and alter their motility (Watson, 1995). Therefore, the researchers have introduced the term "cryocapacitation" to emphasize the fact that cryopreservation procedures induce capacitation-like changes in spermatozoa (Bailey et al., 2000; Cormier & Bailey, 2003; Watson, 1995). These cooling-related capacitation-like changes in spermatozoa, may affect the fertility of cryopreserved semen, by rendering the cells less stable in the reproductive tract, after artificial insemination and therefore relatively short-lived. Such changes cannot easily be distinguished from true capacitation, but Green & Watson (2001) were able to establish that the capacitation-like changes in pig spermatozoa differed from true capacitation in the pattern of tyrosine phosphorylation of proteins.

An increase in both, plasma membrane phospholipid scrambling and phospholipid disorder, during capacitation is associated with enhanced plasma membrane fluidity (Gadella & Harrison, 2002). During freeze–thaw cycle, the sperm membranes undergo lipid phase transition that also leads to an increased disorder of phospholipid packing and membrane fluidity, which causes poor control of intracellular calcium concentration (Bailey & Buhr, 1994; Holt, 2000). Therefore, an alternative stain for assessment of capacitation status of spermatozoa is the hydrophobic probe Merocyanine 540 (M540). This stain detects a decreased packing order of phospholipids in the outer leaflet of the plasma membrane lipid bilayer. Due to the fact that M540 earlier detects changes in the membrane fluidity than CTC, therefore, the hydrophobic probe is believed to be better for evaluating the early events of capacitation (Rathi et al., 2001).
