Cryodamages and their evaluations

More than 50% of all spermatozoa are damaged by freezing process (Leibo 2006).

Formation of ice crystals and the osmotic stress present during freezing and again at thawing are the two major factors related to cryoinjury. However, in recent years a number of other factors related to cryodamage have been characterized: phase transitions in the plasmalemma, oxidative damage, DNA damage, toxicity of cryoprotectants, premature aging, and capacitation-like changes (Watson, 2000). An apoptosis-like phenomenon has been also identified (Anzar et al. 2002, Martin et al.

2004). In general, the most susceptible structures of the spermatozoa to preservation procedures seem to be the membranes (Parks and Graham, 1992). Cryopreserved mammalian semen is generally acknowledged to have an impaired fertility by comparison with fresh semen. The reduction arises from both a lower viability post-thaw and sublethal dysfunction in a proportion of the surviving subpopulation.

Mebrane damages– viability

Changes in plasma membrane structure and integrity appear to be an important component associated with reduced fertility of frozen–thawed spermatozoa. Sperm membrane destabilization occurs when the membrane undergoes a phase transition

from the fluid phase to the gel phase as temperatures decrease (Squires 2005). There are numerous light- and fluorescence microscopic staining methods to determine viability of spermatozoa. These mainly give information from the integrity of the head and acrosome membranes, but not from the tail membrane. The HOS test is thought to have an advantage over the sperm viability stains because it is not only indicative of whether the plasma membrane is intact but also an indicator of whether it is osmotically active (Colenbrander et al. 2003). Neild et al. (2003) have used various fluorochromes to evaluate membrane damages during the freeze-thaw process for equine sperm. Sperm viability and capacitation state were simultaneously evaluated using chlortetracycline (CTC) and Hoechst 33258 dye. Membrane function was also evaluated using HOST. Sperm were analysed immediately after collection, after dilution and centrifugation, after re-dilution and equilibration at room temperature, after cooling to 5 °C, after super-cooling to −15 °C and after thawing. The results show that freezing-thawing induces cell damage and a relative increase in live capacitated/acrosome reacted cells. The most pronounced functional damage to membranes of sperm occurred after thawing. An unique aspect of this study was the ability to evaluate capacitation and acrosomal integrity in conjunction with viability, however only the sperm head membranes were possible to assess. The trypan blue (TB)-neutral red-Giemsa staining method was applied for simultaneous evaluation of sperm head and tail membrane integrity, acrosome status, and overall morphology (Kovács and Foote 1992, Nagy et al. 1999, Kovács et al. 2000). After freezing and thawing, a high proportion of spermatozoa with intact head membranes but damaged tails are observed. These cells are considered as immotile (Nagy et al. 1999).

Decrease in motility

Post-thaw motility of cryopreserved stallion sperm shows poor correlation with fertility, indicating that subcellular damage can affect fertility without concomitant impact on motility. Decline of motility shows species- and individual differences after cryopreservation of spermatozoa. In the semen of the 30–40% of stallions which

‘freeze well’, post-thaw sperm progressive motility and total motility figures of 40–

60% and >70% respectively, but in the semen of 30–40% of stallions that ‘freeze badly’, postthaw sperm progressive motilities as low as 10–15% occur commonly (Jasko et al. 1992, Allen 2005). In the study of Ortega-Ferrusola et al. (2009) a shift from a more linear to a less linear pattern of movement and a significant drop in sperm velocities was observed, rather than a dramatic loss in sperm motility after thawing.

DNA fragmentation

DNA damage is another well-known cytopathic effect of ROS. In equine sperm, exposure to increasing concentrations of ROS resulted in a dose-dependent increase in DNA fragmentation as detected by the Comet Assay. This DNA damage was blocked in the presence of catalase or reduced glutathione (GSH) but not in the presence of SOD, which indicates that hydrogen peroxide (H₂O₂) was the major ROS responsible for DNA damage in these cells (Baumber et al. 2003a). During storage of equine spermatozoa, there is a measurable increase in DNA fragmentation as detected by the comet assay with both cooled (Linfor and Meyers 2002) and frozen storage (Baumber et al. 2003a). In contrast there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation evaluated by sperm chromatin dispersion test (SCD). However, within 1 h of incubation at 37 °C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6 h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage (López-Fernández et al. 2007).

Unfortunately, the addition of antioxidants (α-tocopherol, reduced glutathione, ascorbic acid) or enzyme scavengers (catalase, superoxide dismutase) to cryopreservation extenders did not reduce the level of DNA fragmentation, did not improve spermatozoal motility, acrosomal integrity, viability, or mitochondrial membrane potential subsequent to freezing and thawing of equine sperm cells.

(Baumber et al. 2005). Spermatozoa have limited or no ability to repair DNA damage, and studies indicate that although fertilization may occur, the rate of subsequent embryonic development is reduced and the rate of early embryonic death is increased in situations in which fertilization is initiated by DNA-damaged sperm (Morris et al.

2002).

Apoptosis

In the recent years an apoptosis-like phenomenon has been identified during the cryopreservation process (Anzar et al. 2002; Martin et al. 2004). This explains not only cellular death but also the different degree of subtle cellular damage that most of the surviving population of spermatozoa experiences after thawing. The major function of mitochondria is supplying cellular energy, but the second major function of the mitochondria is the regulation of cell death (Ott et al. 2007). In addition, this subcellular structure is the major source of reactive oxygen species. Mitochondria-generated ROS play an important role in the release of cytochrome C and other proapoptotic proteins, which can trigger caspase activation and apoptosis. In relation to this, mitochondria have been identified as the most sensitive sperm structure to

cryopreservation (Peña et al. 2003). All of these changes result in reduced longevity of the cryopreserved spermatozoa within the female reproductive tract. The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure (Ortega-Ferrusola et al. 2008). Ortega-Ferrusola et al. (2009) studied in equine semen the value of these apoptotic markers as predictor of sperm freezeability. Their findings show differences in the expression of apoptotic markers among stallions; moreover in fresh semen these differences were also observed. After cryopreservation frequently shown morphologic changes in the sperm midpiece that is characterized by moderate to marked swelling of the mitochondria suggesting that sperm mitochondria are a significant site of cryodamage with uncoupling of normal oxidative metabolism, generation of ROS and induction of degenerative processes such as apoptosis (Brum et al. 2008).

Capacitation-like changes, early acrosome lost

Following cryopreservation in modified Kenney's medium, capacitation-like changes were observed evaluated by chlortetracycline (CTC) fluorescence staining. There was a significant increase in the proportion of spermatozoa displaying “capacitated”

pattern (64.8%) and acrosome reacted (AR) pattern (32.8%) with a corresponding decrease in the proportion of spermatozoa displaying the “uncapacitated” pattern (2.5%). There was a major decrease in the proportion of uncapacitated spermatozoa corresponding to an increase of capacitated spermatozoa following removal of seminal plasma after centrifugation and resuspension in freezing medium. (Schembri et al.

2002). Neild et al. (2003) found that freeze-thawing induces cell damage and a relative increase in live/capacitated and live/acrosome reacted cells. However, it was not possible to determine whether the changing CTC patterns reflect a true capacitation phenomenon or an intermediate destabilized state of the sperm cell membrane. In another study (Kavak et al. 2003a) a very low percentage of the cells showed early capacitation sign after thawing detected Merocyanine 540/Yo-Pro-1 probe using flow cytometry. Wilhelm et al. (1996) has used PI and phycoerythrin-conjugated PSA lectin for evaluation of acrosomal status of frozen-thawed stallion spermatozoa, and found that 87–88% of live spermatozoa had intact acrosomes.

Cryopreserved and capacitated sperm share several characteristics such as plasma membrane reorganization, increased intracellular calcium levels, generation of reactive oxygen species, and acquisition of fertilization capacity (Bailey et al. 2000).

Seminal plasma of man and stallion contains cholesterol-rich vesicules secreted by

prostate (prostasome) which block cholesterol efflux from the membrane thus delay the capacitation until appropriate time (Cross and Mahasreshti 1997). Removal of seminal plasma and herewith prostasomes by centrifugation before freezing may influence this physiological process. Generation of reactive oxygen species can promote equine sperm capacitation and tyrosine phosphorylation, suggesting a physiological role for ROS generation by equine spermatozoa (Baumber et al. 2003b).

Thomas et al. (2006) found that the regulation of phospholipid scrambling, the capacitation-like alterations in the plasma membrane and protein tyrosine phosphorylation following cryopreservation are not identical to those in in vitro capacitated equine spermatozoa thus capacitation and ‘‘cryocapacitation’’ are not equivalent processes.

Morphology

After freeezing and thawing, ultrastructural changes were observed in the acrosome, in the outer fibres of the midpiece, and in the axoneme of the principal piece (Christensen et al. 1995, Katila 2001a). Results of SEM showed spermatozoa with typical fenestrations and ruptures of the plasma membrane in the acrosomal region, some spermatozoa with abnormal necks and some specimens with frequently separated head and flagella. Large areas of rough or disrupted acrosomal surface and with ruffled membranes occurred in frozen samples (Blottner et al. 2001).

Functional changes, in vitro „fertility ability” tests: Binding to oviductal epithelial cells and zona pellucida

Spermatozoa that have been altered during the process of freezing and thawing have a reduction in their ability to attach to the oviductal epithelial cells (OEC), hence making a smaller reservoir in the mare’s reproductive tract (Lefebvre and Samper 1993, Dobrinski et al. 1995, Samper 2001). The mean number of spermatozoa bound to equine OEC and zona pellucida (ZP) and percentage of acrosome-intact spermatozoa were lower for frozen-thawed than for fresh spermatozoa. The motility of spermatozoa attached to OEC was lower in cocultures of OEC with frozen-thawed spermatozoa than with fresh spermatozoa at each time point between 0.5 and 48 hours, probably reflecting lower sperm motility in the insemination dose (Dobrinski et al. 1995).

Early embryonic loss

Many authors suggest that frozen–thawed spermatozoa are associated with an increased incidence of early embryonic mortality. The potential mechanisms can now

be studied more effectively. DNA damage may indicate functional damage to the nuclear structures. Possible importance of sperm RNA to the events before the embryonic genome is activated cannot be disregarded (Watson 2000).

Cryodamages, laboratory assessment methods and their correlation to the frozen equine sperm’s fertility

The results of the studies evaluating correlation of frozen equine sperm quality parameters and fertility are contradictory. The fertility of frozen semen is influenced by a number of factors including semen quality, stallion selection, freezing technique, insemination dose, mare selection, mare status and management. The fertility of frozen semen in commercial programmes has been reported to range between 32%

and 73% per cycle (Samper 2001) and between 56% and 89% per season (Loomis 2001). Katila (2001a) has extensively reviewed the different techniques available to assess sperm quality in stallion. Her primary conclusion was that sperm motility and viability were the best parameters. However, within the same research group, a review by Kuisma et al. (2006) later claimed that the fertility of frozen-thawed semen samples from stallions was unpredictable using current laboratory methodologies.

It’s not easy to find correlation between fertility of frozen stallion semen and laboratory tests. It is difficult and expensive to inseminate an adequate number of mares to achieve statistically significant differences. In the early studies there were contradictory results from relation of frozen/thawed equine sperm evaluation methods and fertility parameters: Significant, but low correlations have been demonstrated between the foaling rate and subjective motility of sperm incubated for 2 h and 4 h at 37°C (Katila et al. 2000b) and hypoosmotic swelling test after 0 and 3 h of incubation (Katila et al. 2000a). Significant correlations have been reported between the pregnancy rate (based on 40 mares) and viability of propidium iodide-stained sperm assessed by flow cytometry (Wilhelm et al. 1996) as well as for glass wool and Sephadex filtration tests (Samper et al. 1991). Using CASA system, motility had a low (0.45) but significant correlation with the first-cycle pregnancy rate of 177 mares inseminated with frozen semen from 9 stallions (Samper et al. 1991). In another study there was no correlation between fertility and subjective post-thaw motility or percentage of sperm moving >30 µm/sec (RAP) analysed by CASA (Bataille et al.

1990). In another French study, 766 mares were inseminated with frozen semen, but none of the criteria measured by CASA (VCL, LIN, ALH, MOT, RAP) had a significant correlation with fertility (Palmer and Magistrini 1992). It is clear that freezing and thawing processes cause sublethal changes, premature cryocapacitation and acrosome reaction of spermatozoa, damage membranes and kill cells. Not all of

these changes are reflected in motility. In spite of that, motility estimation by light microscope is the most commonly used method to evaluate frozen-thawed stallion sperm.

Analysis of single-sperm parameters was not highly correlated with stallion fertility in the experiment of Wilhelm et al. (1996). However, with a statistical model that included data on percentage of viable sperm (flow cytometric estimates measured by PI-PSA), percentage of motile spermatozoa and percentage of hamster oocytes penetrated, these tests were highly correlated with stallion fertility /r = 0.85; P = 0.002/ (Wilhelm et al. 1996). Kuisma et al. (2006) detected both negative and positive correlations between HOST and fertility, suggesting that this test is not suitable for evaluation of frozen-thawed stallion semen. In their experiment plasma membrane integrity with light microscopy correlated with many other parameters, including motility. This is in disagreement with the study of Samper (1992) who noted membrane integrity to show extremely poor correlation with motility, particularly in preserved semen. HOST combined with eosin stain for evaluation membrane physical and functional integrity and trypan blue-Giemsa staining for evaluation of sperm plasma membrane and acrosome integrity together was a valuable fertility predictive test could be used for the prognosis of the potential fertility of frozen-thawed bovine semen samples used for IVF or AI according to Tartaglione and Ritta (2004). In the study of Kirk et al. (2005) evaluating only a single parameter did not adequately explain differences among stallions in fertility, however, combining results of assays that measured multiple sperm attributes improved the ability to evaluate the fertilizing potential of frozen-thawed spermatozoa. The four-variable model, which included: (1) motility at 90 min; (2) straightness measured by CASA at 90 min; (3) percentage of live cells evaluated by flow cytometry using propidium iodide and SYBR-14.; and (4) mitochondrial membrane potential measured by flow cytometry, using mitochondrial probe, JC-1, explained the majority of the variation in first cycle fertility between stallions (r² = 0.93) (Kirk et al. 2005). The ultimate goal of multi-parametric sperm analysis was to be able to distinguish sperm samples that have potentially good fertilizing potential from those likely to have poor fertility. Katila (2001a) and Colenbrander et al. (2003) also emphasized the need to combine several tests for fertility evaluation of frozen-thawed stallion semen.

„Sperm cryobiology is still a puzzle” (Leibo 2006). Future attempts to optimize sperm cryopreservation may be more useful if attention is paid to the individual characteristics of males instead of pooled sperm specimens of a species (Leibo 2006).