**7.3 Sperm viability**

146 Current Frontiers in Cryopreservation

pellucida, activate the acrosome reaction and eventually fertilize the oocyte (Yanagimachi, 1994). Normally, capacitation *in vivo* occurs in the female reproductive tract, but capacitation can also be induced in vitro by the incubation in capacitating media which the most of media contain the bicarbonate, calcium and serum albumin (Yanagimachi, 1994). Furthermore, It has been demonstrated that the cooling and freezing process can also induced the capacitation like change which affect to the low fertilizing capacity of spermatozoa in boar and other mammalian species (Maxwell and Johnson, 1997; Green and Watson, 2001; Barrios et al., 2000). During cooling, freezing and rewarming process, it is hypothesized that change in low temperature cause the modification and destabilization of the lipid content in the sperm plasma membrane, reducing the selective permeability resulted in the cholesterol efflux and intracellular calcium uptake leading to the capacitation like change (Green and Watson, 2001; Tardif et al., 2001). To improve the the FT spermatozoa, there are some studies about the addition of cholesterol-loaded cyclodextrins increased the cryosurvival of boar, ram and bovine spermatozoa because cyclodextrins used to deliver cholesterol to the sperm plasma membrane which against cold shock (Purdy and Graham, 2004; Bailey et al., 2008; Mocé et al., 2009). In addition, the supplement of antioxidants such as vitamin E or alpha-tocopherol decreased the capacitation like change of cryopreserved boar spermatozoa (Satorre et al, 2007). Furthermore, the addition of seminal plasma to boar spermatozoa has been shown to reduce the capacitated spermatozoa in chilled and FT boar semen (Kaneto et al., 2002; Suzuki et al., 2002; Vadnais et al., 2005a,b;

Sperm concentration will be assessed by direct cell count using a Bürker haemocytometer (Boeco, Humburg, Germany) (Beardon and Fuquay, 1997). The visual progressive motility of both fresh and FT sperm is evaluated at 38C under a phase contrast microscope at 200x and 400x magnification. The motility is assessed by the same person throughout the

The motility patterns of diluted FT semen are assessed using the CASA system (Halminton Thorne Biosciences IVOS, Version 12 TOX IVOS, Beverly, USA). Each FT thawed semen samples is diluted with pre-warmed Modena extender (37°C) to obtain a final concentration of 50x106 spermatozoa/ml. A 5 µl of diluted semen is pipetted into the chamber and allowed the 1 min before analysis fore sample distribution and pre-warming (Iguer-Ouada and Verstegen, 2001). After the first assessment (T0), the diluted semen is evaluated after incubation at 37 C for 30 min (T30) and 60 min (T60). The camera will recognize the position of the sperm heads in successive frames. Spermatozoa heads are marked with a different color to enable the observer and the analyzer to differentiate between the different motility patterns. Each semen sample is measured twice, 3 fields are evaluated and counted at least 1000 cells per analysis. Motility patterns including (1) Curvilinear velocity (VCL, µm/s), the average velocity measured in the progression line along the whole track of cell

Okazaki et al., 2009, Garcia et al., 2009).

experiment.

**7. Laboratory method for semen quality assessment** 

**7.1 Sperm concentration and progressive motility** 

**7.2 Computer-assisted sperm analysis (CASA)** 

The percentages of sperm viability will be determined by 2 methods. The first one is eosinnigrosin staining (Dott and Foster, 1972). The semen sample (50 µl) are mixed well with a drop of eosin-nigrosin dyes (Fluka Chemie GmbH, Sigma-Aldrich, Switzerland), and the mixture (10 μl) is smeared and dried on a glass slide. Evaluation is undertaken by counting 200 spermatozoa with 1000x magnification. Spermatozoa with an unstained head are regarded as live spermatozoa. The second method is evaluated by SYBR-14/Ethidiumhomodimer-1 (EthD-1) (Fertilight®, Sperm Viability Kit, Molecular Probes Europe, Leiden, The Netherlands). This technique is modified after Axnér et al. 2004 and Garner and Johnson, 1995). Ten µl of diluted semen are mixed with 2.7 µl of the user solution of SYBR-14 and 10 µl of EthD-1. The user solution is SYBR-14 diluted (1:100) in dimethyl sulfoxide (DMSO), fractionated and frozen in eppendorfs. After incubation at 37 C for 20 min, two hundred spermatozoa will be assessed (x1000) under fluorescent microscope. The nuclei of the spermatozoa with an intact plasma membrane are stained green with SYBR-14, while those with damaged membranes stained red with EthD-1.

Fig. 3. Spermatozoa stained with SYBR-14/EthD-1 or PI: live spermatozoa stained green with SYBR-14 while dead spermatozoa stained red with EthD-1 or PI.

Cryopreservation of Boar Spermatozoa: An Important Role of Antioxidants 149

Fig. 4. Sperm DNA damage (black circle) stained with a commercial kit (Halomax staining)

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".

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

**7.7 Chlortetracycline (CTC) assay** 

**7.8 Annexin-V/PI assay** 

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 percentage of live spermatozoa with intact plasma membranes.
