**7.7 Chlortetracycline (CTC) assay**

148 Current Frontiers in Cryopreservation

Spermatozoa are classified into three types; live spermatozoa stained green with SYBR-14, dead spermatozoa stained red with EthD-1 and moribund spermatozoa stained both green and red (Axnér et al. 2004; Garner and Johnson, 1995).The results are expressed as the

Acrosome integrity will be assessed using fluorescein isothiocyanate–labeled peanut (*Arachis hypogaea*) agglutinin (FITC-PNA) staining. Ten µl of the diluted semen is mixed with 10 µl of Ethidiumhomodimer-1 and incubated at 37 ºC for 15 min. Five µl of the mixture is smeared on a glass slide and fixed with 95 % ethanol for 30 second. Fifty µl Fit C-PNA (dilute Fit C-PNA with PBS 1:10 v/v) is spread over the slide and incubated in a moist chamber at 4 ºC for 30 min. After incubation, it is rinsed with cold PBS and air dried. Two hundred spermatozoa are assessed under fluorescent microscope at 1000x magnification and classified as intact acrosome, damaged acrosome and missing acrosome (Cheng et al., 1996; Axner et al., 2004). The results are scored as the percentage of intact acrosome

The functional integrity of the sperm plasma membrane will be assessed using a short hypoosmotic swelling test (sHOST) (Perez-Llano et al., 2001). Spermatozoa are incubated, at 38 C for 30 min, with 75 mOsm/kg a hypo-osmotic solution that consist of 0.368 % (w/v) Nacitrate and 0.675 % (w/v) fructose (Merck, Germany) in distilled water. Following this incubation time, 200 µl of the semen-hypo-osmotic solution is fixed in 1000 µl of a hypoosmotic solution plus 5 % formaldehyde (Merck, Germany), for later evaluation. Two hundred spermatozoa are assessed under a phase contrast microscope at 400x magnification. The coiled tail (sHOST positive) spermatozoa found following incubation are

DNA damage can be evaluated by Acridine orange (AO) staining or Halomax staining method (Fig. 4). The technique is modified after Thuwanut et al. (2008). Briefly, two smears from each sample were prepared on glass slide and air-dried. Each smear is fixed overnight in Carnoy's solution, freshly prepared with methanol and glacial acetic acid (3:1 v/v). The slide is removed from the fixative solution, air-dried, and then stained with 1% (100 mg/ml) AO (Sigma) in distilled water for 10 min. The AO staining solution is prepared by adding 10 ml of 1% AO in distilled water to 40 mL of 0.1 M citric acid (Merck, Darmstadt, Germany) and 2.5 ml of 0.3 M Na2HPO4.7H2O (Merck, Darmstadt, Germany) pH 2.5. The AO staining solution will be prepared daily and stored in the dark at room temperature until use. After staining, the slide is gently washed by distilled water and covered with the cover slip. One thousand spermatozoa are evaluated under the fluorescence microscope. The heads of the sperm cells with normal DNA (double-stranded) have green fluorescence, while those with damaged or single stranded DNA showed orange or red fluorescence. The results are expressed as the proportion of the damage/single stranded DNA per 1,000 counted

percentage of live spermatozoa with intact plasma membranes.

**7.5 The functional integrity of the sperm plasma membrane** 

**7.4 Acrosome integrity** 

spermatozoa.

**7.6 DNA damage** 

spermatozoa.

functional intact plasma membrane.

The CTC assay is slightly modified from as described previously (Harayama et al., 2000). The CTC staining solution containing 750 μm CTC, 5 mM DL-cysteine, 130 mM NaCl and 20mM Tris (hydroxymethyl aminomethane) (pH 7.8) is prepared immediately before use. This solution is protected from light until analysis. Briefly, 50 μl of sperm suspension will be mixed with 50 μl of CTC staining solution for 30 sec, followed by the addition of 10μl 12.5% paraformaldehyde in 0.5 M Tris-HCl (pH 7.4) as a fixative. Then, 10 μl of the sperm suspension is mixed well with equal volume of antifade solution (0.22 M of 1,4 diazabicyclo[2,2,2]octane ;DABCO) in glycerol:PBS (9:1) on the microscopic slide and gently compressed with coverslip. Two slides are prepared from each sample and stored in the dark at at 4 °C until evaluation. Two hundred spermatozoa per slide will be under a Nikon fluorescence microscope at 400x under blue-violet illumination (excitation at 400–440 nm and emission at 470 nm). The spermatozoa are classified in to three staining patterns described by Fraser et al. (1995). F-pattern, fluorescence over the whole region of the sperm head are considered to be "non-capacitated spermatozoa". B-pattern, fluorescence in the acrosomal region except post-acrosomal region are considered to be "capacitated spermatozoa". AR-pattern, low or no fluorescence over the whole head except thin bright ban in the equatorial segment are considered to be "acrosome-reacted spermatozoa".

#### **7.8 Annexin-V/PI assay**

Apoptosis will be evaluated by apoptosis detection kit ApopNexin™ (Chemicon Int., USA) using a fluorescent microscope. This assay will detect the phosphatidylserine translocation

Cryopreservation of Boar Spermatozoa: An Important Role of Antioxidants 151

docosahexanoyl (22:6) groups which both lipids represent approximately 50 to 60 % of total phospholipids in boar and bull spermatozoa (Pursel and Graham, 1967; Johnson et al., 1969; Parks and Lynch, 1992). Cholesterol was the major sterol in sperm lipids of all species. Cholesterol to phospholipid molar ratios were 0.26, 0.30, 0.36, and 0.45 for sperm plasma membrane of the boar, rooster, stallion, and bull, respectively (Parks and Lynch,

The susceptibility of spermatozoa to cold shock differ among species because of the differences of lipid composition of the sperm plasma membrane among species (Flesch and Gadella, 2000).The resistance of the mammalian spermatozoa to cold shock was high in species in which the cholesterol to phospholipids molar ratio and the phospholipids saturation is high (Darin-Bennett and White, 1977). The avian spermatozoa have a high level of cold shock resistant and have a higher level of saturated phospholipids compared to mammalian sperm (Parks and Lynch, 1992). The plasma membrane of the boar spermatozoa is characterized by a high protein, low cholesterol and high proportion of EP compared to other species (Parks and Lynch, 1992; Nikolopoulou et al., 1985). In contrast, the protein content and EP proportion of rooster sperm plasma membrane is low while the cholesterol

As mentioned above the sperm plasma membrane has a very high amounts of polyunsaturated fatty acids (PUFAs) especially docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) (Johnson et al., 1969; Parks and Lynch, 1992). It has been suggested that the proportion of unsaturated fatty acid influence the properties of sperm plasma membrane (Miller et al., 2005). High levels of long chain PUFAs, DPA and DHA, are associated with an increased membrane fluidity (Quinn, 1985). During cryopreservation, the fluidity of the plasma membrane from boar spermatozoa is significantly decreased when compared to fresh spermatozoa which tend to restrict the post-thawed sperm quality (Buhr et al., 1994). In human, sperm with a high level of membrane fluidity had a higher postthawed motility compared to sperm with a low level of membrane fluidity after

Docosahexaenoic acid (commonly known as DHA; 22:6 (n-3)) is an omega-3 essential polyunsaturated fatty acid. DHA is most often found in cold water fatty fish (salmon fish, tuna fish) and in fish oil supplements, along with eicosapentaenoic acid (EPA). DHA is the main fatty acid composition of the spermatozoa as well as the brain and the retina (Neuringer et al., 1988). For the sperm plasma membrane, DHA play a major role in regulating membrane fluidity in sperm and in the regulation of spermatogenesis (Haidl and Opper, 1997; Ollero et al., 2000). DHA content is significantly higher in immature

Studies have demonstrated that the supplement of PUFAs in the feed of the boar improve the quality of the boar spermatozoa (Paulenz et al., 1999; Rooke et al., 2001; Strezezek et al., 2004 ;Maldjian et al., 2005). In addition, Rooke et al. (2001) found that tuna oil supplemented in the boar diet increase viability, progressive motility and normal morphology. The supplementation of PUFAs also enhanced the survival rate of post-thawed boar spermatozoa (Strezezek et al., 2004). DHA improved the reproductive performance of the male turkey (Blesbois et al., 2004). Maldjian et al. (2005) found that the use of DHA-enriched

1992).Glycolipids represented less than 10% of total polar lipids for all species.

content is intermediate (Parks and Lynch, 1992).

cryopreservation (Giraud et al., 2000).

**9. Docosahexaenoic acid (DHA)** 

spermatozoa than mature spermatozoa.

from inner to outer leaflet of cell plasma membrane which is the hallmark of apoptosis during the degradation phase. Following manufacturer instructions, sperm cells are washed twice with PBS (pH 7.4) by centrifugation at 400 g for 5 min. Sperm pellet are resuspended with HEPES buffer (10mM HEPES/NaOH, pH7.4, 150mM NaCl, 5mM KCl, 1mM MgCl2, 1.8mM CaCl2, 2x106 sperm/ml). One hundred l of sperm suspension are mixed well with 5 l annexinV-FITC conjugate and 3 l of Propidium iodide (PI;20g/ml) and incubated for 15 min at room temperature in the dark. Two hundred spermatozoa are assessed under fluorescent microscope at 400x magnification. The apoptotic sperm cells will fluorescence green while necrotic sperm cells fluorescence red. Alternatively, flow cytometry analysis can also be used instead of fluorescent microscope (Fig. 5).

Fig. 5. FCS/SCC two-dimensional histogram, flow cytometry analysis of frozen-thawed boar spermatozoa (supplemented with L-cysteine) stained with Annexin-V/PI: Q3 represent the viable spermatozoa with intact plasma membrane, while Q2 represent dead spermatozoa with non-intact plasma membrane
