**3.2.1 Sperm membrane integrity**

The integrity of sperm membranes is a necessary condition to maintain spermatozoal functions during storage in the female's reproductive tract and penetration of the oocyte (Holt, 2000). When semen is frozen, cells are exposed to a cold shock, ice crystals formation, and cellular dehydratation, which all cause irreversible damage (Amann, 1999; Parks & Graham, 1992). Cellular membranes are one of the primary sites of injury during chilling, freezing and thawing. Damage is caused by alteration of membrane structure and lateral organization (Amann, 1999). The cryopreservation results in temperature-dependent and dehydratation-induced membrane phase changes, which are thought to result in lateral phase separation of membrane components and increased membrane permeability for solutes (Hammerstedt et al., 1990). The disruption of plasma membrane integrity caused by disarrangement of lipids within the membrane during cryopreservation may induce further cellular damage and consequently lead to a sperm death (Watson, 1995).

Membrane integrity of mammalian and avian spermatozoa may be assessed by using many fluorescent probe combinations including: carboxyfluorescein diacetate (CFDA) in combination with propidium iodide (PI), SYBR-14 with PI, carboxy-seminaphthorhodfluor (Carboxy-SNARF) with PI, calcein-AM with ethidium homodimer (EthD-1) and Hoechst

Methods of Assessment of Cryopreserved Semen 557

ethidium homodimer allowing for simultaneous assessment of acrosomal status and

The motility of spermatozoa subjected to cryopreservation is reduced by reason of some changes in the active transport and the permeability of the plasma membrane in the tail region (Blesbois et al., 2008; Watson, 1995). A reduction of spermatozoa motility may also be triggered by a change in the availability of energy or an injury of the axonemal elements. Moreover, it has been noted that the alterations in the ultrastructure of mitochondria occurring during cryopreservation are followed by a loss of the internal mitochondrial

Rhodamine 123 (R123) is the potentiometric membrane dye which is used to selectively stain functional mitochondria. It fluoresces only when the proton gradient over the inner mitochondrial membrane (IMM) is built up and unstained sperm do not contain functional mitochondria (Garner et al., 1997; Gravance et al., 2001). Also the group of Mitotrackers: Mitotracker Deep Red, Red, Orange and Green selectively label the respirating mitochondria. Thus, these probes are suitable to discriminate sperm with deteriorated mitochondria from sperm in which oxidative respiration occurs (Gadella & Harrison, 2002;

Some of mitotrackers such as 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) change their fluorescent properties due to changes in the potential of IMM. JC-1 is a lipophilic cationic fluorescent carbocyanine dye that is internalized by all functioning mitochondria, where it fluoresces green. However, as the concentration of JC-1 inside the mitochondria increases (highly functional mitochondria), the stain forms aggregates which fluoresce orange. Hereby, population of spermatozoa can be divided into high (orange staining), moderate (orange and green) and low (green) mitochondrial potential groups after IMM depolarisation (Fig. 6c) (Garner et al. 1999;

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

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,

membrane integrity (Fig. 6b) (Kawakami et al., 1993; Szász et al., 2000).

structure of frozen-thawed spermatozoa (Watson, 1995).

**3.2.3 Mitochondrial function** 

Garner et al., 1997).

Gravance et al., 2000).

**3.2.4 Capacitation status** 

not been developed in birds (Lemoine et al., 2008).

33258 (Christensen et al., 2004; Donoghue et al., 1995; Hewitt & England, 1998; Partyka et al., 2010; Peña et al., 1998; Rijsselaere et al, 2005; Sirivaidyapong et al., 2000). SYBR-14 and CFDA, usually used detectors of live cells, are membrane-permeant and non-fluorescent compounds, which are immediately deacylated and thus rapidly converted into high fluorescent compounds by intracellular esterases. These green fluorochromes are maintained intracellular by intact membranes (Peña et al., 1998; Silva & Gadella, 2006). As plasma membrane deteriorate at cell death, cells lose their ability to resist the influx of red fluorescent PI. PI replaces or quenches green fluorochromes (Garner & Johnson, 1995). Live, viable, intact spermatozoa show a green fluorescence (CFDA, SYBR-14, calcein-AM) while dead stain red (PI) (Fig. 6a). Carboxy-SNARF is pH indicator which stains live spermatozoa orange, while bisbenzimide stain Hoechst 33258 labels dead spermatozoa bright blue (Hewitt & England, 1998). The last one requires flow cytometric analysis with a laser that operates in the ultraviolet light range and is less commonly used in andrology laboratory, however alternatively it may be applied within fluorescent microscope.

SYBR-14/PI fluorochromes have been found to be more sensitive in comparison with conventional method of live-dead cell assessment. The advantage of the use of fluorochromes is the possibility to assess the semen without the interference of fat particles and others material present in the extended semen (Rijsselaere et al., 2005). The detection of the third subpopulation i.e. moribund spermatozoa is the next advantage of this method. Additionally the simultaneous assessment of several functions of spermatozoa may be done in the same specimen by simultaneous staining of sperm cells with fluorescent lectins PNA or PSA for acrosome evaluation with PI for dead cell assessment.
