**2.2 Sperm morphology**

On account of the fact that freezing and thawing process provokes morphological or biochemical cryogenic damage resulting in sperm dysfunction and changes in cell's membrane, the sperm morphology evaluation is an essential component of any semen analysis and provides the clinical information about the potential fertility of semen sample.

Despite, there are many different and also new methods, as described below, used in semen analysis, semen smears are still employed for routine light microscopic morphological evaluation. However, this assessment is subjective and results are largely dependent on the proficiency and experience of the evaluator. Vital dye in combination with different stains for acrosome evaluation are commonly utilised to assess the spermatozoa morphology and the viability together. For this purpose, India ink, William's, Karras, Spermac, Diff-Quick, Papanicolaou, Fuelgen or combination: Trypan blue and Giemsa, Trypan blue, Bismarck Brown and Rose Bengal, and finally eosin-nigrosin (described in 2.3 section) have been used in birds and mammals including human (Brito et al., 2003; Brito et al., 2011; Didion et al., 1989; Freneau et al., 2010; Łukaszewicz et al., 2008; Partyka et al., 2007; Rodriguez-Gil et al., 1994; Sprecher & Coe, 1996; Talbot & Chacon, 1981). In spite of that, Freneau et al. (2010) have shown that differential interference phase contrast microscopy of wet-mounted semen is the superior method for bulls sperm morphology assessment. For cats sperm morphology, the best differentiation of sperm structures, especially acrosome, with lower artifacts, fast green FCF-rose Bengal staining or Hancock and Glendhill solution staining and phasecontrast microscope are encouraged (Zambelli & Cunto, 2006). However, when frozenthawed semen is analyzed these stains are negatively affected by egg yolk and glycerol, causing egg yolk agglutination and lack of sperm structures differentiation. Therefore, sperm washing is recommended to prevent these interferences (Zambelli & Cunto, 2006).

Many reports have shown the common classification system for the morphology of spermatozoa from different species. However, classification categories are different for the various species and the adoption of uniform system within each species is needed. Mammalian spermatozoa abnormalities can be divided into primary and secondary abnormalities (Blom, 1950), or in some classification systems into major and minor abnormalities (Blom, 1968, 1983). Primary sperm defects are assumed to have occurred during spermatogenesis, and secondary defects are assumed to have occurred during maturation in the epididymis and the transit through the ductal system and specimen preparation. Second system classifies sperm defects according to the perceived effects on fertility. The most common sperm abnormalities (Fig. 1) are related to abnormal acrosomal regions/heads, detached head, proximal droplets, distal droplets, abnormal midpieces, bent/coiled tails. Acrosome defects include knobbed, roughed, and detached acrosomes. Head defects include microcephalic, macrocephalic, pyriform, tapered, other shape defects, nuclear vacuoles, and multiple heads. Midpiece and principal piece (tail) abnormalities enclose simple bent, folded, fractured, thickened, swollen, roughed, Dag-like, disrupted sheet, duplicated, coiled. Various defects are typical for each species.

For each slide, at least 100-300 spermatozoa should be counted at 400-1000x magnification, which allows for accurate calculation of the percentage of different sperm defects.

of motile spermatozoa does not require expensive equipment, is a simple and rapid method for assessment of sperm quality, however, it is a highly subjective and not reliable assay for

On account of the fact that freezing and thawing process provokes morphological or biochemical cryogenic damage resulting in sperm dysfunction and changes in cell's membrane, the sperm morphology evaluation is an essential component of any semen analysis and provides the clinical information about the potential fertility of semen sample. Despite, there are many different and also new methods, as described below, used in semen analysis, semen smears are still employed for routine light microscopic morphological evaluation. However, this assessment is subjective and results are largely dependent on the proficiency and experience of the evaluator. Vital dye in combination with different stains for acrosome evaluation are commonly utilised to assess the spermatozoa morphology and the viability together. For this purpose, India ink, William's, Karras, Spermac, Diff-Quick, Papanicolaou, Fuelgen or combination: Trypan blue and Giemsa, Trypan blue, Bismarck Brown and Rose Bengal, and finally eosin-nigrosin (described in 2.3 section) have been used in birds and mammals including human (Brito et al., 2003; Brito et al., 2011; Didion et al., 1989; Freneau et al., 2010; Łukaszewicz et al., 2008; Partyka et al., 2007; Rodriguez-Gil et al., 1994; Sprecher & Coe, 1996; Talbot & Chacon, 1981). In spite of that, Freneau et al. (2010) have shown that differential interference phase contrast microscopy of wet-mounted semen is the superior method for bulls sperm morphology assessment. For cats sperm morphology, the best differentiation of sperm structures, especially acrosome, with lower artifacts, fast green FCF-rose Bengal staining or Hancock and Glendhill solution staining and phasecontrast microscope are encouraged (Zambelli & Cunto, 2006). However, when frozenthawed semen is analyzed these stains are negatively affected by egg yolk and glycerol, causing egg yolk agglutination and lack of sperm structures differentiation. Therefore, sperm washing is recommended to prevent these interferences (Zambelli & Cunto, 2006). Many reports have shown the common classification system for the morphology of spermatozoa from different species. However, classification categories are different for the various species and the adoption of uniform system within each species is needed. Mammalian spermatozoa abnormalities can be divided into primary and secondary abnormalities (Blom, 1950), or in some classification systems into major and minor abnormalities (Blom, 1968, 1983). Primary sperm defects are assumed to have occurred during spermatogenesis, and secondary defects are assumed to have occurred during maturation in the epididymis and the transit through the ductal system and specimen preparation. Second system classifies sperm defects according to the perceived effects on fertility. The most common sperm abnormalities (Fig. 1) are related to abnormal acrosomal regions/heads, detached head, proximal droplets, distal droplets, abnormal midpieces, bent/coiled tails. Acrosome defects include knobbed, roughed, and detached acrosomes. Head defects include microcephalic, macrocephalic, pyriform, tapered, other shape defects, nuclear vacuoles, and multiple heads. Midpiece and principal piece (tail) abnormalities enclose simple bent, folded, fractured, thickened, swollen, roughed, Dag-like, disrupted

sheet, duplicated, coiled. Various defects are typical for each species.

For each slide, at least 100-300 spermatozoa should be counted at 400-1000x magnification,

which allows for accurate calculation of the percentage of different sperm defects.

the prediction of fertility (Peña Martínez, 2004).

**2.2 Sperm morphology** 

Fig. 1. Selected defects in sperm morphology (boar spermatozoa): a) normal sperm cells; b) looped tail; c) acrosome detachment; d) loss of acrosomal contents (back arrow), proximal cytoplasmatic droplet (arrowhead); e) proximal cytoplasmatic droplet; f) kinked midpiece; g) looped tail (black arrow), coiled tail (arrowhead); h) thickened midpiece.

Methods of Assessment of Cryopreserved Semen 551

Recently, computer assisted sperm analysis has been introduced to veterinary andrology, same as it has been used in reproductive technologies in human andrology (Rijsselaere et al., 2003; Verstegen et al., 2002). This technique assures objective semen assessment, whereas the main disadvantage of conventional semen evaluation is variability of obtained results. Subjectivity of traditional semen analysis is associated mainly with experience and skill of the observer, the method of specimen preparation, staining technique and number of cells evaluated. Variations in the results of conventional evaluation of the same semen samples by different observers and laboratories may achieve up to 30-60% (Coetzee et al., 1999; Davis & Katz, 1992). Subsequently, correlations between spermatozoa characteristics and fertility trials in females are relatively low. Computer assisted sperm analysers allow for calculation of several motility parameters, which characterize movement of individual sperm cells. They include VAP-average path velocity, VSL-straight line velocity, VCL-cell velocity, ALHamplitude of lateral head displacement, BCF-beat cross frequency (Fig. 4), STR-straightness of cell track, LIN-linearity of cell track, subpopulation of rapid, medium and slow cells (Niżański et al., 2009). Selected characteristics of spermatozoa motility parameters measured

Fig. 4. Scheme of different velocities and parameters of sperm movement measured by

Fig. 3. HOS test (canine spermatozoa).

**3. Advanced methods of semen assessment 3.1 Computer assisted sperm analysis (CASA)** 

by CASA systems are summarized in table 1.

CASA systems.

## **2.3 Sperm membrane integrity**

**Live-dead staining.** The traditional method for assessing whether the sperm membrane is intact or disrupted involves examining a percentage of viable sperm by a stain exclusion assay. For the determination of cell viability live-dead stains as aniline-eosin, eosin-nigrosin or eosin-fast green are widely used. Integrity of the plasma membrane is shown by the ability of a viable cell to exclude the dye, whereas the dye will diffuse passively into sperm cells with damaged plasma membranes. When stained smears are viewed under the oil immersion objective of light microscope, the percentage of viable, live, properly formed spermatozoa, nonviable and also partially-damaged spermatozoa can be determined. In eosin-nigrosin stain under the microscope, live spermatozoa appear white, unstained against the purple background of nigrosin (Fig. 2a). Dead and damaged spermatozoa which have a permeable plasma membrane are pink (Fig. 2b). The evaluation of the percentage of live and dead spermatozoa and the percentage of morphology defects may be performed on the same nigrosin-eosin stained slides.

Fig. 2. Eosin-nigrosin staining for live-dead cells (chicken spermatozoa): a) live spermatozoon, b) dead spermatozoa.

**The hypoosmotic swelling test (HOS)** is a method of investigating membrane integrity in sperm and, as such, is an alternative to supra-vital staining. In fact, the HOS test is thought to have the advantage of indicating not only whether the membrane is intact, but also whether it is osmotically active. Sperm with an intact, functional membrane when are exposed to an hypoosmotic solution incubated for 30 minutes at 37ºC, swell to achieve an osmotic equilibrium. An expression of this is a typical swelling of the sperm tail (Fig. 3) (Neild et al., 1999). The HOS test is a simple, inexpensive and easily applicable technique, which has been adapted to assess spermatozoa of several species (Corea & Zavos, 1994; Kumi-Diaka, 1993 Neild et al., 1999; Pérez-Llano et al., 2001; Santiago-Moreno et al., 2009). It has been suggested that this test may supplement the information provided by the conventional parameters of semen analysis, and is useful for fertilizing ability assessment (Brito et al., 2003; Vazquez et al., 1997). This test correlates highly with other predictive tests, such as hamster oocyte penetration (Jeyendran et al., 1992), in-vitro fertilization (IVF) results in human (van der Venn et al., 1986), and with pregnancy rates in pigs (Pérez-Llano et al., 2001). The HOS test seems to be more appropriate for predicting the fertilizing capacity of frozen-thawed than fresh semen, because membrane damage is here a more important limiting factor than in the former (Colenbrander et al., 2003).

**Live-dead staining.** The traditional method for assessing whether the sperm membrane is intact or disrupted involves examining a percentage of viable sperm by a stain exclusion assay. For the determination of cell viability live-dead stains as aniline-eosin, eosin-nigrosin or eosin-fast green are widely used. Integrity of the plasma membrane is shown by the ability of a viable cell to exclude the dye, whereas the dye will diffuse passively into sperm cells with damaged plasma membranes. When stained smears are viewed under the oil immersion objective of light microscope, the percentage of viable, live, properly formed spermatozoa, nonviable and also partially-damaged spermatozoa can be determined. In eosin-nigrosin stain under the microscope, live spermatozoa appear white, unstained against the purple background of nigrosin (Fig. 2a). Dead and damaged spermatozoa which have a permeable plasma membrane are pink (Fig. 2b). The evaluation of the percentage of live and dead spermatozoa and the percentage of morphology defects may be performed on

Fig. 2. Eosin-nigrosin staining for live-dead cells (chicken spermatozoa): a) live

limiting factor than in the former (Colenbrander et al., 2003).

**The hypoosmotic swelling test (HOS)** is a method of investigating membrane integrity in sperm and, as such, is an alternative to supra-vital staining. In fact, the HOS test is thought to have the advantage of indicating not only whether the membrane is intact, but also whether it is osmotically active. Sperm with an intact, functional membrane when are exposed to an hypoosmotic solution incubated for 30 minutes at 37ºC, swell to achieve an osmotic equilibrium. An expression of this is a typical swelling of the sperm tail (Fig. 3) (Neild et al., 1999). The HOS test is a simple, inexpensive and easily applicable technique, which has been adapted to assess spermatozoa of several species (Corea & Zavos, 1994; Kumi-Diaka, 1993 Neild et al., 1999; Pérez-Llano et al., 2001; Santiago-Moreno et al., 2009). It has been suggested that this test may supplement the information provided by the conventional parameters of semen analysis, and is useful for fertilizing ability assessment (Brito et al., 2003; Vazquez et al., 1997). This test correlates highly with other predictive tests, such as hamster oocyte penetration (Jeyendran et al., 1992), in-vitro fertilization (IVF) results in human (van der Venn et al., 1986), and with pregnancy rates in pigs (Pérez-Llano et al., 2001). The HOS test seems to be more appropriate for predicting the fertilizing capacity of frozen-thawed than fresh semen, because membrane damage is here a more important

**2.3 Sperm membrane integrity** 

the same nigrosin-eosin stained slides.

a) b)

spermatozoon, b) dead spermatozoa.

Fig. 3. HOS test (canine spermatozoa).

## **3. Advanced methods of semen assessment**

#### **3.1 Computer assisted sperm analysis (CASA)**

Recently, computer assisted sperm analysis has been introduced to veterinary andrology, same as it has been used in reproductive technologies in human andrology (Rijsselaere et al., 2003; Verstegen et al., 2002). This technique assures objective semen assessment, whereas the main disadvantage of conventional semen evaluation is variability of obtained results. Subjectivity of traditional semen analysis is associated mainly with experience and skill of the observer, the method of specimen preparation, staining technique and number of cells evaluated. Variations in the results of conventional evaluation of the same semen samples by different observers and laboratories may achieve up to 30-60% (Coetzee et al., 1999; Davis & Katz, 1992). Subsequently, correlations between spermatozoa characteristics and fertility trials in females are relatively low. Computer assisted sperm analysers allow for calculation of several motility parameters, which characterize movement of individual sperm cells. They include VAP-average path velocity, VSL-straight line velocity, VCL-cell velocity, ALHamplitude of lateral head displacement, BCF-beat cross frequency (Fig. 4), STR-straightness of cell track, LIN-linearity of cell track, subpopulation of rapid, medium and slow cells (Niżański et al., 2009). Selected characteristics of spermatozoa motility parameters measured by CASA systems are summarized in table 1.

Fig. 4. Scheme of different velocities and parameters of sperm movement measured by CASA systems.

Methods of Assessment of Cryopreserved Semen 553

(Davis & Katz, 1992; Iguer-Ouada & Verstegen, 2002; Rijsselaere et al., 2003; Verstegen et al., 2002). Also other factors as the type and depth of the used chamber, number of fields analysed, temperature during analysis and protocol of semen sample preparation affect results. Optimization and validation of the technical settings would allow to compare intraand inter-laboratory results, regardless of the instruments that have been used (Agarwal et

Computer assisted sperm analysis allows for a detailed estimation of subtle changes of sperm motion characteristics such as hyperactivation (HA) of spermatozoa associated with capacitation process. Hyperactivation is the process that mammalian spermatozoa exhibit, while they progress through the female oviduct. It is described as vigorous, nonprogressive, non-linear sperm motion linked with capacitation. During HA, the pattern of sperm track undergo dramatic changes, characterized by wide-amplitude marked lateral movements of the head and tail of the spermatozoon, with slow or non-progressive 'starpin' movement (Verstegen et al., 2002). Hyperactivated sperm movement, is assumed to be necessary, for mammalian sperms to penetrate into and pass through, the cumulus cell layer of an oocyte (Meyers et al., 1997; Suarez et al., 1983). To fertilize the oocyte, mammalian spermatozoa must be capacitated, the process that depends on the removal or alteration of substances absorbed on, or integrated in the sperm plasma membrane, resulting in changes in membrane permeability and intracellular ionic composition, with Ca2+ movements playing the most critical role (Fraser et al., 1995; Rota et al., 1999). ALH and velocity parameters such as path velocity VAP, progressive velocity VSL are increased in hyperactivated spermatozoa, whereas linearity LIN and straightness STR of movement are lowered. Such changes are characteristic for capacitation induced by specific media (Rota et al., 1999) and for spermatozoa that underwent preservation (cryocapacitation) and are pronounced, especially when media with addition of detergents are used (Niżański et al., 2009). Kawakami et al. (2001) observed that oviduct's epithelium possess the ability to bind hyperactivated spermatozoa, which results in the obvious prolongation of their flagellar movement. On the other hand, the life-span of the free moving non-bound hyperactivated spermatozoa within oviductal lumen, is relatively shorter. It was also found, that Ca influx into the cytoplasm is inhibited in the oviductepithelium-binding sperms (Dobrinsky et al., 1997). Active movement of the sperms and Ca influx into cytoplasm negatively affect the maintenance of viability and fertile life of sperm in the lumen of oviduct. Binding to the oviduct epithelium presumably prevents Ca influx, required for sperm capacitation. This phenomenon is available for prolonging viability and fertile life of canine sperms in the oviduct (Kawakami et al., 2001). Considering the obvious lack of such regulatory mechanism, in frozen-thawed semen it is believed, that in vitro post-thaw hyperactivation results in depletion in spermatozoa energy resources, accumulation of metabolites in the extender and cell death, if insemination dose is not deposited into the female's genital tract immediately after

Nevertheless, the computer assisted sperm analysis of cryopreserved semen should be treated with a dose of criticism. It should be emphasized, that CASA parameters describing kinematic features of frozen-thawed sperm cells may not reflect the real loss of quality of ejaculate after treatment. Absolute CASA parameters (VCL, VSL, VAP, ALH, BCF) should be used with caution, whereas relative CASA parameters (combinations of absolute

al., 1992).

thawing.


Table 1. Selected parameters of spermatozoa motility measured by CASA systems.

It was proven in human, that results obtained with CASA systems are better correlated with the outcome of assisted reproductive techniques than results of traditional semen evaluation (Verstegen et al., 2002). Blesbois et al. (2008) showed that some of parameters detected in CASA system are correlated with fertility results obtained with frozen–thawed chicken spermatozoa (PMOT, PROG, VAP, VSL). Most of them were affected by cryopreservation, with the exception of straightness (STR), suggesting that cryopreservation slows down the movement of chicken spermatozoa without changing the shape of trajectories.

The important advantage of computer assisted sperm analysers is the immediate measurement of sperm concentration, total number of sperms in ejaculate and the automated calculation of number of insemination units which could be prepared from one ejaculate. Additionally some machines are equipped with UV excitation module, which gives the opportunity to analyse the percentage of live and dead spermatozoa after staining with vital fluorescent probes, such as Hoechst 33258. Nevertheless, CASA system needs standardization and validation before its use and image settings have been standardized

minimum speed as determined by values defined under setup.

**MOT** % Motility - The population of cells that are moving at or above a

**PMOT** % Progressive motility- the population of cells that are moving actively

**VCL** µm/s Track speed – Is defined as average velocity measured over the actual point-to-point track followed by the cell.

**VAP** µm/s Path velocity - Is defined as average velocity over smoothed average

**BCF** Hz Beat Cross Frequency – is the frequency with which the sperm head moves back and forth in its track across the cell path.

**STR** % Straightness - A measure of VCL side to side movement determined

**LIN** % Linearity - A measure of the departure of the cell track from a straight

**VSL** µm/s Progressive velocity - Is measured in the straight line distance between the beginning and the end of the track. **ALH** µm Amplitude of Lateral Head Displacement – is the mean width of the

head oscillation as the cell moves.

**Parameter Unit Description** 

forward.

position of the cell.

by the ratio VSL/VAP.

**RAP** % Rapid – subpopulation of rapid cells.

**SLOW** % Slow – subpopulation of slow cells.

**STATIC** % Static cells.

the shape of trajectories.

line. It is the ratio VSL/VCL.

**MED** % Medium – subpopulation of cells with medium velocity.

Table 1. Selected parameters of spermatozoa motility measured by CASA systems.

It was proven in human, that results obtained with CASA systems are better correlated with the outcome of assisted reproductive techniques than results of traditional semen evaluation (Verstegen et al., 2002). Blesbois et al. (2008) showed that some of parameters detected in CASA system are correlated with fertility results obtained with frozen–thawed chicken spermatozoa (PMOT, PROG, VAP, VSL). Most of them were affected by cryopreservation, with the exception of straightness (STR), suggesting that cryopreservation slows down the movement of chicken spermatozoa without changing

The important advantage of computer assisted sperm analysers is the immediate measurement of sperm concentration, total number of sperms in ejaculate and the automated calculation of number of insemination units which could be prepared from one ejaculate. Additionally some machines are equipped with UV excitation module, which gives the opportunity to analyse the percentage of live and dead spermatozoa after staining with vital fluorescent probes, such as Hoechst 33258. Nevertheless, CASA system needs standardization and validation before its use and image settings have been standardized (Davis & Katz, 1992; Iguer-Ouada & Verstegen, 2002; Rijsselaere et al., 2003; Verstegen et al., 2002). Also other factors as the type and depth of the used chamber, number of fields analysed, temperature during analysis and protocol of semen sample preparation affect results. Optimization and validation of the technical settings would allow to compare intraand inter-laboratory results, regardless of the instruments that have been used (Agarwal et al., 1992).

Computer assisted sperm analysis allows for a detailed estimation of subtle changes of sperm motion characteristics such as hyperactivation (HA) of spermatozoa associated with capacitation process. Hyperactivation is the process that mammalian spermatozoa exhibit, while they progress through the female oviduct. It is described as vigorous, nonprogressive, non-linear sperm motion linked with capacitation. During HA, the pattern of sperm track undergo dramatic changes, characterized by wide-amplitude marked lateral movements of the head and tail of the spermatozoon, with slow or non-progressive 'starpin' movement (Verstegen et al., 2002). Hyperactivated sperm movement, is assumed to be necessary, for mammalian sperms to penetrate into and pass through, the cumulus cell layer of an oocyte (Meyers et al., 1997; Suarez et al., 1983). To fertilize the oocyte, mammalian spermatozoa must be capacitated, the process that depends on the removal or alteration of substances absorbed on, or integrated in the sperm plasma membrane, resulting in changes in membrane permeability and intracellular ionic composition, with Ca2+ movements playing the most critical role (Fraser et al., 1995; Rota et al., 1999). ALH and velocity parameters such as path velocity VAP, progressive velocity VSL are increased in hyperactivated spermatozoa, whereas linearity LIN and straightness STR of movement are lowered. Such changes are characteristic for capacitation induced by specific media (Rota et al., 1999) and for spermatozoa that underwent preservation (cryocapacitation) and are pronounced, especially when media with addition of detergents are used (Niżański et al., 2009). Kawakami et al. (2001) observed that oviduct's epithelium possess the ability to bind hyperactivated spermatozoa, which results in the obvious prolongation of their flagellar movement. On the other hand, the life-span of the free moving non-bound hyperactivated spermatozoa within oviductal lumen, is relatively shorter. It was also found, that Ca influx into the cytoplasm is inhibited in the oviductepithelium-binding sperms (Dobrinsky et al., 1997). Active movement of the sperms and Ca influx into cytoplasm negatively affect the maintenance of viability and fertile life of sperm in the lumen of oviduct. Binding to the oviduct epithelium presumably prevents Ca influx, required for sperm capacitation. This phenomenon is available for prolonging viability and fertile life of canine sperms in the oviduct (Kawakami et al., 2001). Considering the obvious lack of such regulatory mechanism, in frozen-thawed semen it is believed, that in vitro post-thaw hyperactivation results in depletion in spermatozoa energy resources, accumulation of metabolites in the extender and cell death, if insemination dose is not deposited into the female's genital tract immediately after thawing.

Nevertheless, the computer assisted sperm analysis of cryopreserved semen should be treated with a dose of criticism. It should be emphasized, that CASA parameters describing kinematic features of frozen-thawed sperm cells may not reflect the real loss of quality of ejaculate after treatment. Absolute CASA parameters (VCL, VSL, VAP, ALH, BCF) should be used with caution, whereas relative CASA parameters (combinations of absolute

Methods of Assessment of Cryopreserved Semen 555

During last decades many fluorescent probes have been used for the semen assessment. The fluorescence of these compounds may be estimated using fluorescent microscopy or flow cytometry. Flow cytometry enables the observation of physical characteristics such as cell size, shape, and also any component or function of the spermatozoon that can be detected by a fluorochrome or fluorescently labeled compound. The analysis is objective and accurate. The great number of spermatozoa (>10 000) can be analyzed in a small volume of samples in a short time. This is considerably more than the total of 200 cells generally observed by microscopic analysis. Thus, the analysis of events detected on dot plots gives the accurate and high reliable results (Peña et al., 2001). It is a sensitive method of detection of subtle differences among spermatozoal populations that may not be detected with other

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

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

cellular damage and consequently lead to a sperm death (Watson, 1995).

Fig. 5. System for Assisted Sperm Morphology Assessment.

**3.2 Flow cytometry and fluorescent probes** 

techniques.

**3.2.1 Sperm membrane integrity** 

features-LIN, STR) can not be used directly for estimation of semen quality. Selective death of the most immotile and weakened spermatozoa leads to the situation, where normal CASA parameters show the 'pseudoenhancement' of kinematics. Thus, the mean velocity and linearity parameters may be higher after freezing. This is caused by the fact, that the sub-population of the most resistant cells which survive freezing-thawing may possess higher mean quality parameters, than the larger population of motile sperm cells in fresh semen. In spite of the fact, that only half or one third of population of sperm cells may survive the cryopreservation, their mean velocity may be higher in comparison with velocity parameters of larger population of spermatozoa in fresh semen. Thus, some investigators (Katkov & Lulat, 2000) observed increase in kinematic parameters (KP) of specimen after freezing-thawing, while at the same time substantial losses in post-thaw motility (percentage of motile cells) were observed. The possible explanation of this phenomenon is the selective elimination of the slowest sub-population within the specimens. This "CASA-paradox" is caused by substantial exclusion of slow-moving cells from the motile fraction measured after freezing-thawing.

Therefore, in order to obtain more reliable results of semen assessment after thawing, it was proposed to use Modified Kinematics Parameters (MKP) or Yield of Kinematic Parameters (YKP). MKP can be defined as KP that is average on an entire sample:

$$\text{MKP} = \text{KP} \times \text{Motion} / 100\% \tag{1}$$

YKP is the product of KP and the number of motile cells for which this parameter is average:

$$\text{YKP} = \text{Total Number of Multile Cells} \times \text{KP} / 100\% \tag{2}$$

Furthermore, morphology (Assisted Sperm Morphology Assessment-ASMA) of sperm cells can be objectively evaluated, on the basis of morphometric analysis of predefined specific measurements of particular elements in spermatozoa. Usually, on the slides, the head morphometric dimensions of length, width, width/length, area and perimeter of a minimum of 200 sperm are analyzed (Fig. 5). Additionally, parameters of head shape can be evaluated such as ellipticity, circularity, elongation, and regularity (Álvarez et al., 2008). Nevertheless, the accuracy of sperm morphology assessment depends on the careful preparation, fixation and staining of spermatozoa. The analysis of sperm morphology may be done using Diff-Quik stain recommended by World Health Organization (WHO, 2010) or SpermBlue, which has been developed for the evaluation of human and animal sperm morphology (Maree et al., 2010).

Rubio-Guillen et al. (2007) showed that by applying ASMA techniques and multivariate cluster analysis, it is possible to determine three subtle subpopulations of spermatozoa with different morphometric characteristics coexisting in bull ejaculates. The proportion of spermatozoa in each sperm subpopulation showed considerable differences among males and varied significantly throughout the cryopreservation procedure. The cryopreservation of spermatozoa has been found to affect chromatin structure and morphometry of the sperm head (Arruda et al., 2002; Esteso et al., 2003; Gravance et al., 1998; Hidalgo et al., 2006; Rijsselaere et al., 2004). Thus, it is presumed that the adverse effects of cryopreservation on sperm chromatin and head morphology, may be responsible for lowered fertility of spermatozoa, observed after cryopreservation.

features-LIN, STR) can not be used directly for estimation of semen quality. Selective death of the most immotile and weakened spermatozoa leads to the situation, where normal CASA parameters show the 'pseudoenhancement' of kinematics. Thus, the mean velocity and linearity parameters may be higher after freezing. This is caused by the fact, that the sub-population of the most resistant cells which survive freezing-thawing may possess higher mean quality parameters, than the larger population of motile sperm cells in fresh semen. In spite of the fact, that only half or one third of population of sperm cells may survive the cryopreservation, their mean velocity may be higher in comparison with velocity parameters of larger population of spermatozoa in fresh semen. Thus, some investigators (Katkov & Lulat, 2000) observed increase in kinematic parameters (KP) of specimen after freezing-thawing, while at the same time substantial losses in post-thaw motility (percentage of motile cells) were observed. The possible explanation of this phenomenon is the selective elimination of the slowest sub-population within the specimens. This "CASA-paradox" is caused by substantial exclusion of slow-moving cells

Therefore, in order to obtain more reliable results of semen assessment after thawing, it was proposed to use Modified Kinematics Parameters (MKP) or Yield of Kinematic Parameters

YKP is the product of KP and the number of motile cells for which this parameter is average:

Furthermore, morphology (Assisted Sperm Morphology Assessment-ASMA) of sperm cells can be objectively evaluated, on the basis of morphometric analysis of predefined specific measurements of particular elements in spermatozoa. Usually, on the slides, the head morphometric dimensions of length, width, width/length, area and perimeter of a minimum of 200 sperm are analyzed (Fig. 5). Additionally, parameters of head shape can be evaluated such as ellipticity, circularity, elongation, and regularity (Álvarez et al., 2008). Nevertheless, the accuracy of sperm morphology assessment depends on the careful preparation, fixation and staining of spermatozoa. The analysis of sperm morphology may be done using Diff-Quik stain recommended by World Health Organization (WHO, 2010) or SpermBlue, which has been developed for the evaluation of human and animal sperm

Rubio-Guillen et al. (2007) showed that by applying ASMA techniques and multivariate cluster analysis, it is possible to determine three subtle subpopulations of spermatozoa with different morphometric characteristics coexisting in bull ejaculates. The proportion of spermatozoa in each sperm subpopulation showed considerable differences among males and varied significantly throughout the cryopreservation procedure. The cryopreservation of spermatozoa has been found to affect chromatin structure and morphometry of the sperm head (Arruda et al., 2002; Esteso et al., 2003; Gravance et al., 1998; Hidalgo et al., 2006; Rijsselaere et al., 2004). Thus, it is presumed that the adverse effects of cryopreservation on sperm chromatin and head morphology, may be responsible for lowered fertility of

MKP = KP x Motility /100% (1)

YKP = Total Number of Motile Cells x KP /100% (2)

from the motile fraction measured after freezing-thawing.

morphology (Maree et al., 2010).

spermatozoa, observed after cryopreservation.

(YKP). MKP can be defined as KP that is average on an entire sample:

Fig. 5. System for Assisted Sperm Morphology Assessment.
