**6. Capacitation like changes of the frozen-thawed boar semen and its influence on** *in vivo* **fertility**

After ejaculation, the spermatozoa are not able to fertilize the oocyte. They undergo the activation process that called ' capacitation' and acrosome reaction which spermatozoa will able to reach the ampulla of the oviduct, penetrate the cumulus oophorus, bind to the zona

Cryopreservation of Boar Spermatozoa: An Important Role of Antioxidants 147

path; (2) Average pathway velocity (VAP, m/s) , the average velocity of the smoothed cell path; (3) Straight line velocity (VSL), the average velocity measured in a straight line from the beginning to the end of the track (m/s); (4) The amplitude of the lateral head displacement (ALH), the mean width of the head oscillation as the sperm cells swim (m); (5) The beat cross-frequency (BCF, Hz), frequency of the sperm head crossing the average path in either direction; (6) The straightness (STR, %) = average value of the ratio VSL/VAP;

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.

(7) The Linearity (LIN, %) = average value of the ratio VCL/VAP.

**7.3 Sperm viability** 

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