Morphology and membrane integrity
- In Vitro Evaluation of Frozen-Thawed Stallion Semen: A Review
In some studies increases in sperm abnormalities have been associated with decreased fertility [7,40], but others have found no relation between morphology of fresh semen and fertility [89,27]. A wide range of morphological deviations may be acceptable for breeding stallions, if the total number of morphologically normal motile spermatozoa in the ejaculate is adequate . Sometimes the low pregnancy rates after frozen semen inseminations are simply due to an excessively small number of live morphologically normal post-thaw sperm. Morphological features are evaluated by light microscope using different sperm stains. The use of fluorescent probes requires epifluorescence optics for the microscope. Scanning and transmission electron microscopic techniques are not in routine use, but have been useful in some abnormal cases and in research. One has to be cautious in the interpretation of transmission images.  noticed that membrane-bound vesicles in acrosomal and midpiece regions are not caused by freezing and thawing. They are damaged during preparation of samples.
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 . Automated morphometric analysis provides accurate objective measurements of sperm head and shape [25,51].
The simplest examination method is to fix sperm cells in buffered formol-saline or buffered glutaraldehyde solution and view unstained cells with either phase-contrast or differential interference-contrast microscopy. General-purpose cellular stains (Wright's, Giemsa, haematoxylin-eosin, India ink) can be used , but live-dead stains (aniline-eosin, eosin-nigrosin, eosin-fast green) are more widely used for the determination of cell viability. 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 . Glycerol can interfere with the staining properties of these dyes making them less reliable for the evaluation of cryopreserved semen . Differential stains for sperm cells are Spermac , William's and Casarett's stains , Triple stain, Papanicolau, and Feulgen and Karras among others . The Spermac stain was not found to be very useful in the evaluation of frozen stallion semen by , although it has been in routine use in Germany . It is generally recommended that 200 cells be examined, but evaluation of 100 sperm cells probably provides a valid representation of abnormalities .
A combination of 2 fluorescent stains, e.g. carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) or calcein AM and ethidium homodimer, can be used to assess cell viability. CFDA and calcein AM molecules cross cell membranes and are de-esterified by esterases within the cell. They are retained within intact cells, causing them to fluoresce green. PI and ethidium homodimer cannot penetrate living cells, but can only bind to and stain cellular DNA in damaged cells, giving them red fluorescence . Other frequently used fluorescent dyes are Hoechst 33258, ethidium bromide (EB) and SYBR14.
The most commonly used method to detect acrosome integrity is staining with fluorescein-conjugated lectins, such as Peanut Agglutinin (PNA), Pisum Sativum Agglutinin (PSA) or Concanavalin A (ConA) coupled with fluoresceinisothiocynate (FITC) [51,10]. FITC-PNA with ethidium homodimer as a counter stain allowed for a rapid and reliable assessment of the acrosomal status of stallion sperm. Acrosome-intact spermatozoa displayed intense green fluorescence over the acrosomal cap, while acrosome-reacting spermatozoa showed a patchy disrupted image of fluorescence. Sperm cells that had completed the acrosome reaction acquired a stain on the equatorial segment or remained unstained . Chlortetracycline assay (CTC) is used to detect capacitation and acrosome reactions of the spermatozoa . Mitochondrial activity can by evaluated by Rhodamine 123 (R123), which is a fluorescent dye used to label a negative potential (the inside of the mitochondria being negative) across the inner mitochondrial membrane. Only coupled, respiring mitochondria will take up this fluorescent dye. A good correlation has been shown between sperm viability and mitochondrial function for equine spermatozoa [17,69].  used another fluorescent dye, JC-1, to assess mitochondrial function in equine sperm. They concluded that JC-1 accurately reflects changes in mitochondrial membrane potential.
Typically, 100 to 400 fluorescent cells are counted under microscope. A fluorometer can be used to evaluate the proportion of fluorescent cells rapidly. This method has been applied to frozen boar sperm  and also to fresh  and frozen stallion semen [43,44]. In our study, frozen-thawed stallion sperm were stained with PI and fluorescence determined; however, no correlation with fertility was established [43,44]. A very rapid and effective method is flow cytometry, which allows thousands of individual cells to be evaluated. Multiple aspects of sperm function can be assayed simultaneously. Sperm viability, DNA content and the proportion of acrosome-reacted sperm can be investigated using this method . The cost of sorting flow cytometry at the moment is very high, and therefore, is not used in routine work.
Fluorescent probes have been used to evaluate different steps of the freezing process , and compare modifications in freezing  or thawing techniques . The dual SYBR-14/PI stain has been used to assess quality of frozen-thawed stallion semen. Live spermatozoa emit green fluorescence (SYBR-14 +), and dead ones emit red colour (PI+). There was a negative correlation (r = -0.49) between the percentage of rapidly moving spermatozoa as estimated by HTM and the percentage of spermatozoa emitting red fluorescence (PI+). In contrast, a positive correlation (r = 0.35) was found between the percentage of rapid sperm and those emitting green fluorescence . Highly significant correlations were seen between MOT (Strömberg-Mika-Cell-Motion-Analysis-System) and intact spermatozoa, when frozen-thawed stallion semen was stained with (CFDA/PI) . Motility of frozen-thawed stallion semen (VCL, MOT and ALH) was significantly correlated with degree of degradation of the plasma membrane as evaluated by FITC-Con-A. Addition of glycerol caused significant reductions in VCL and ALH and increased the proportion of damaged spermatozoa, but the most pronounced changes in motility were observed after freezing and thawing . In another study, FITC-PSA with ethidium homodimer as a counter-stain was used to evaluate acrosomal status of stallion semen. Freezing and thawing resulted in a high percentage of acrosome-reacted or -damaged sperm and a significant decrease in sperm viability, suggesting an enhanced level of sperm capacitation-like changes or membrane damage . When stallion semen samples with known percentages of acrosome-damaged spermatozoa were incubated with PSA, a positive correlation (0.98) was found between the percentage of spermatozoa bound to PSA and the percentage of acrosome-damaged spermatozoa .
Studies on integrity of plasma and acrosomal membranes of frozen-thawed sperm have increased in the past years. It remains to be seen how well membrane integrity correlates with fertility results. Flow cytometric evaluation of viability of frozen-thawed PI-stained stallion (5 stallions) spermatozoa correlated with the fertility (r = 0.68) of 40 mares (80 cycles), and was better (p .
Monoclonal antibodies and indirect immunolabelling techniques
A primary antibody specific for an acrosomal antigen can be used to evaluate integrity of acrosomal membranes. The antigen is localized at the inner surface of the outer acrosomal membrane. Only cells with damaged plasma and acrosomal membranes will bind primary antibody and demonstrate fluorescence after exposure to a secondary antibody (anti-mouse IgG-FITC) when viewed by epifluorescence microscopy. In a German study, Spermac and immunohistochemical staining with monoclonal antibody were compared in the evaluation of acrosomes of frozen-thawed stallion sperm. Significantly more damaged acrosomes were diagnosed by Spermac (31%) as compared with monoclonal antibody (25%) .  concluded that immunohistochemical staining with monoclonal antibody was superior to conventional staining techniques (Spermac and Karras) in assessing acrosomal status of frozen stallion semen.
Hypo-osmotic swelling test (HOS)
When spermatozoa are suspended in a hypo-osmotic solution, water will enter the spermatozoon in an attempt to attain osmotic equilibrium. This increases the volume of the cell, thereby reducing the initial length of the flagellum, and the plasma membrane bulges . The influx of water only occurs in the tail region and creates different types of curls. The appearance of a curl in the tail of a sperm is a sign that water has been transported in a physiological manner into the cell to reach osmotic equilibrium. This indicates an intact flagellar membrane .
 recommended that 100 μl of stallion semen is added to 1 ml of 100 mOsm sucrose solution and incubated at 37°C for 60 min. They found the test to be simple, accurate and consistent with good reliability and repeatability.  saw vesicles in stallion sperm tails most frequently, when the osmolality was between 150 and 100 mOsm and  between 100 and 25 mOsm. When testing 156 ejaculates from 13 stallions, a significant positive correlation was obtained between HOS and fertilization rate .
Resistance of stallion spermatozoa to hyperosmotic stress (600 to 4000 mOsm) was not useful in the evaluation of frozen-thawed stallion semen . Several semen evaluation methods were applied in the assessment of fresh and frozen stallion semen in a French study. HOS was performed on fresh semen immediately after collection, and after an incubation of 4 h and 6 h at 37°C in the presence or absence of seminal plasma. After freezing and thawing, the HOS-test was carried out at 0 h and after an incubation of 4 h at 37°C, and after a storage of 7 days at 4°C. The HOS-test applied immediately after semen collection was highly correlated with MOT and ATP levels after thawing (r > 50) at 0 and 4 h and with MOT after 7 days. The authors suggested that HOS applied after collection of fresh semen is the best predictive test of the freezability of stallion semen .  tested commercially used frozen semen from 31 stallions and compared results with foaling rates of 1085 mares. The HOS-test was carried out using a 100 mOsm solution and an incubation of 45 min at 37°C. A significant correlation was found between foaling rate and HOS-test performed on sperm immediately after thawing or after an incubation of 3 h at 37°C.
When stallion sperm (fresh, freeze-damaged, uterine-inoculated) were filtrated through cotton, glass wool (GW) and Sephadex (S) filters, the results indicated that spermatozoa with acrosome-damaged or -reacted sperm were trapped by GW filters. Spermatozoa with capacitation-like changes (uterine-inoculated sperm) were trapped by S-filters . In filtration of frozen-thawed semen of 9 stallions, significant correlations were obtained between the pregnancy rate per cycle (177 mares) and the percentage of sperm passing through the filters (GWS, r = 0.93 and S, r = 0.84) . If Sephadex traps capacitated spermatozoa, this finding would indicate that capacitation of spermatozoa is a problem with frozen-thawed sperm. GW-filtered human spermatozoa showed an increased capability to penetrate zona-free hamster oocytes . Motility did not account for the improved penetrability. When the filtered spermatozoa were diluted with nonviable spermatozoa, the improved oocyte penetration disappeared. Thus, it was concluded that the removal of nonviable spermatozoa may, at least, in part, be responsible for this effect . The results of  and  look promising, but, so far, filtration tests have not gained widespread acceptance.  considered GWS-filtration to be unreliable in the evaluation of frozen-thawed stallion semen, because 75% of the variance was due to error (straws, tubes, ejaculates). However, this statement was not substantiated with fertility results.
Cells with membrane damage lose essential metabolites and enzymes. Numerous enzymes have been determined in semen of several species, most often bulls and boars. These include aspartate-aminotransferase (AT-ase), fumarase, isocitratedehydrogenase, aconitase, arylsulphatases (AS), Na+/K+-ATPase, glutamic oxaloacetic transaminase (GOT), lactic dehydrogenase (LDH), cholinesterase, acid phosphatase and alkaline phosphatase [13,75]. AS-ases are present in the acrosome of the intact sperm cell and in seminal plasma. Membrane damage to the mid-piece results in release of AT-ase to the seminal plasma. As a result, ATP production is blocked, immobilising the sperm cell .  has advocated the use of AT-ase as a good predictor of stallion semen freezability, suggesting that the higher the enzyme levels, the lower the motility after thawing. However, this was neither statistically analysed nor substantiated by fertility trials.
Acrosin is a proteolytic enzyme present in the acrosome and thought to be important in acrosome reaction, sperm-zona binding and zona penetration.  determined acrosin amidase activity from raw semen, from semen extended in freezing extender and from frozen-thawed stallion semen. Acrosin activity increased with sperm concentration (r2 = 0.75, p .  identified acrosin activity in stallion semen before and after freezing by means of a gelatine substrate method. Acrosin activity was detected by the presence of halos around single sperm, resulting from localized proteolytic digestion of gelatin. Morphological alterations of the acrosome and acrosin activity were correlated (r = 0.9, p .
GOT is an intracellular enzyme with limited usefulness due to its presence in high concentrations in cytoplasmic droplets . After freezing and thawing of boar semen, a heterospermic index was correlated with the following in vitro tests: spermatozoa with acrosin-activity (0.38), extracellular GOT (0.54), intracellular GOT (-0.57) and motility (0.50) at 7 h post-thaw . The authors pointed out that the extracellular GOT present immediately following ejaculation should be determined along with the GOT following freezing and thawing. The prefreeze GOT-values are then subtracted from post-thaw GOT-values because boars differ greatly in extracellular GOT before freezing.
The intact sperm cell has a relatively high content of ATP. If membranes are defective, the nucleotide phosphates will leak out of the cell into the seminal plasma and be hydrolyzed. ATP/ADP/AMP measurements in stallion sperm provide information on membrane viability . Intracellular ATP content reflects mitochondrial activity of the stallion spermatozoon and can be determined by bioluminescence . In their study, ATP and HOS were correlated shortly after semen collection, after 6 h survival at 37°C and after 4 h survival post-thaw at 37°C. The integrity of the plasma membrane of the flagellum seems to be essential for maintaining the mitochondrial activity and the ATP content . In fresh and frozen stallion semen, ATP content was correlated with objective motility (r = 0.92) and velocity (r = 0.87) (p . The ATP content of the frozen-thawed stallion sperm was reduced 50% from the concentration in fresh semen .
Determinations of enzyme concentrations in semen have been practised for a long time. They are simple, rapid and inexpensive to do. On the other hand, they are prone to errors. It is necessary to select an enzyme found only in sperm cells. In addition to spermatozoa, enzymes can be present in cytoplasmic droplets, seminal plasma, and organic extenders. No convincing results have yet been presented that would favour the use of enzyme determinations in assessing pre- and post-thaw semen quality.
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