**5. Harmful effect of cryopreservation to spermatozoa**

The use of FT boar semen under field conditions results in low conception rate and reduced number of total piglets born per litter (Eriksson et al, 2002). These problems occur because of the poor post-thawed semen quality and low survival rate of boar spermatozoa after cryopreservation (Hammerstedt et al., 1990; Curry et al., 2000). The detrimental effects of cooling, freezing and thawing caused subsequently impaired the membrane integrity, structure and function of the spermatozoa, eventually fertilizing ability (Hammerstedt et al., 1990; Guthrie and Welch, 2005).

It is well documented that the boar spermatozoa are highly susceptible to temperatures below 15 C. The viability of the spermatozoa are dramatically reduced within a few hours after expose to cooling below 15 C, so call 'cold shock' (Gilmore et al., 1996). Cold shock caused the damage of plasma membranes and alterations in the metabolism of the spermatozoa. This caused by changes in the arrangement of plasma membrane compositions especially phospholipids (reviewed by Medeiros et al., 2002).The sperm damage cause by cold shock is characterized by an irreversible loss of motility and the loss of sperm permeability. Boar spermatozoa seem to acquire a cold shock resistance when the

Shortly after collection, the semen is diluted (1:1 v/v) with extender I (Modena™, Swine Genetics International, Ltd., Iowa, USA). The diluted semen is transferred to 50 ml centrifuge tubes, equilibrated at 15 ºC for 120 min and centrifuged at 800x g for 10 min. The supernatant is discarded and the sperm pellet was re-suspended (about 1-2:1) with extender II (80 ml of 11% lactose solution and 20 ml egg yolk) to a concentration of 1.5x109 spermatozoa/ml. The diluted semen is cooled to 5 ºC for 90 min. Then, two parts of the semen are mixed with one part of extender III (89.5% of extender II with 9% glycerol and 1.5% Equex-STM®). The final concentration of semen is approximately 1.0x109 spermatozoa/ml and contained 3% glycerol (modified after modified after Westerndorf et al., 1975 and Gadea et al., 2004). The processed semen is loaded into 0.5 ml straws (Bio-Vet, Z.I. Le Berdoulet, France). The straws are sealed with PVC powder before being placed in contact with nitrogen vapour about 3 cm above the liquid nitrogen level for 20 minutes in an expandable polystyrene box. Then the straws are plunged into liquid nitrogen (-196 ºC) for storage. Thawing is achieved by immersing the straws in water at 50C for 12 sec (Selles et al., 2003). Immediately after thawing, the semen is diluted (1:4) with a Modena™ extender. Post-thawed sperm qualities are evaluated after incubation in a 37C water-bath for 15 min.

After incubation in extender I, the semen is divided into 4 groups according to the composition of extender II. Group I, extender II containing 80 ml of 11% lactose solution and 20 ml egg yolk. Group II, extender II is supplemented with 0.29 g of fish oil (Fish oil 1000; Blackmores LTD, New Southwell, Australia; containing DHA 120 mg g fish oil) per gram of egg yolk. Normal egg yolk contains approximately 3.15 mg DHA per gram of egg yolk, as was analyzed at the Institute of Nutrition, Mahidol University (AOAC, 2007). Group III is supplemented with a combination of fish oil 0.29 g and L-cysteine 5 mM (Fluka Chemie GmbH, Sigma-Aldrich, Switzerland). Group IV is supplemented with a combination of fish

The use of FT boar semen under field conditions results in low conception rate and reduced number of total piglets born per litter (Eriksson et al, 2002). These problems occur because of the poor post-thawed semen quality and low survival rate of boar spermatozoa after cryopreservation (Hammerstedt et al., 1990; Curry et al., 2000). The detrimental effects of cooling, freezing and thawing caused subsequently impaired the membrane integrity, structure and function of the spermatozoa, eventually fertilizing ability (Hammerstedt et al.,

It is well documented that the boar spermatozoa are highly susceptible to temperatures below 15 C. The viability of the spermatozoa are dramatically reduced within a few hours after expose to cooling below 15 C, so call 'cold shock' (Gilmore et al., 1996). Cold shock caused the damage of plasma membranes and alterations in the metabolism of the spermatozoa. This caused by changes in the arrangement of plasma membrane compositions especially phospholipids (reviewed by Medeiros et al., 2002).The sperm damage cause by cold shock is characterized by an irreversible loss of motility and the loss of sperm permeability. Boar spermatozoa seem to acquire a cold shock resistance when the

**4.2 Semen freezing and thawing procedures** 

**4.3 Semen extender** 

oil 0.29 g and L-cysteine 10 mM.

1990; Guthrie and Welch, 2005).

**5. Harmful effect of cryopreservation to spermatozoa** 

semen is held at room temperature in seminal plasma for 1-5 hours (Pursel et al., 1972). It was found that viability and fertilizing ability of the boar spermatozoa was significantly improved when the fresh semen was held at 15 C for over 3 hours before cryopresenvation (Almlid and Johnson, 1988; Eriksson et al., 2001)

During the process of freezing, the decrease of temperature from -15 C to -60 C causes sperm damage (Mazur, 1985). This causes by the intracellular ice formation and cellular dehydration (osmotic stress). The subsequent physical events depend on the cooling rates. Intracellur ice formation occur during a rapid cooling when intracellular water does not leave the cell to maintain equilibration. If cooling is slow, the spermatozoa will lose water rapidly avoid to intracellular ice formation. However, if spermatozoa are cooled too slowly, they will expose to high concentration of solutes which caused intracellular water to diffuse out of the cell, dehydration both the cell and plasma membrane ( also known as solution effects) (Mazur, 1970; Parks and Graham, 1992). Gilmore et al. (1996) demonstrated that the boar spermatozoa are sensitive to osmotic stress. Similar findings were also found in dog (Songsasen et al., 2002), cat (Pukazhenthi et al., 2000), ram (Curry and Watson, 1994), stallion (Ball and Vo, 2001) and bull (Liu and Foote, 1998).

Cellular damage due to intracellular ice formation and dehydration, oxidative stress is another important cause of sperm damage leading to abnormal sperm structure and function and subfertility. Currently, several studies have been reported oxidative stress affect the damage of sperm membrane, proteins and DNA in human (Agarwal et al., 2003), stallion (Baumber et al., 2000; 2003), bull (Bilodeau et al., 2001) and boar (Roca et al., 2004; 2005)(Fig. 2).

Fig. 2. Scanning electron microscopic (SEM) picture of fresh boar semen with normal plasma membrane (A) as compare with SEM picture of frozen boar semen with plasma membrane damage (B).
