**4.1 Semen collection**

Three ejaculates from each boar are collected using gloved-hand method. During collection the semen is filtered through gauze and only sperm rich fractions are collected. Within 30 min after collection, semen volume, pH, sperm motility, concentration, percentage of live and dead sperm and morphology are determined. Only ejaculates with motility of ≥70% and ≥80 % morphologically normal are used for cryopreservation.

(Almlid and Hofmo, 1996; Johnson, 1998). The widespread exchange of genetic material between breeding populations with liquid stored semen is difficult because of the short life span of the spermatozoa (Wagner and Thibier, 2000; Johnson et al., 2000). The first FT boar spermatozoa have been reported since 1956 (Polge, 1956). Unfortunately, the FT spermatozoa has a very low fertilizing ability. In 1970, the first pregnancy was achieved with FT boar semen using a surgical insemination technique (Polge et al., 1970). In 1971, many studies have reported the pregnancies after intra-cervical insemination using FT boar

In general, there are many important factors in the process of FT boar semen that affect the post-thawed semen quality. For instance, the semen collection technique, equilibration time, type of semen extender, type and concentration of cryoprotectant, freezing package, freezing rate and thawing procedure (Johnson et al., 2000). Many types of freezing package have been used for frozen boar semen, such as medium straws, maxi straws (Bwanga et al., 1991; Berger and Fisherleitner, 1992), plastic bags (Bwanga et al., 1991) and FlatPack®/MiniFlatPack® (Eriksson and Rodriguez-Martinez, 2000). Most containers has been developed for suitable storage, transport, post thawed semen quality and practical

The freezing and thawing procedure have a significant impact on the survival rate of sperm after cryopreservation (Johnson et al., 2000). However, optimal freezing and thawing rates vary depending on the type and concentration of the cryoprotectant (Mazur et al., 1970; Fiser et al., 1993). Currently, the optimal rates for boar sperm freezing appear to be 30C/min with 3% glycerol as cryoprotectant when freezing in 0.5 ml straws (Fiser et al., 1990) and 16C/min with 3.3% glycerol in 5 ml straws (Pursel et al., 1985). For both these methods the optimal thawing rate is 1200 C/min (Westerndorf et al., 1975; Fiser et al., 1993). Eriksson and Rodriguez-Martinez, (2000) found that the optimal freezing rate was 50 C/min in 3% glycerol with a 900 C/min thawing rate for flattened plastic bags (FlatPack®) container. A variety of cryoprotectants are used in the freezing extender of different in species. Glycerol, egg yolk and sodium dodecyl sulphate (SDS) (Equex STM or Orvus ES paste) is commonly used as cryoprotectants for the cryopreservation of boar semen (Westerndorf et al., 1975; Pursel et al., 1978; Holt, 2000b). The optimal concentration of glycerol was approximately 3 % in pig (Holt, 2000a). Egg yolk and SDS are non-permeable cryoprotectant used in freezing extender and provide protective effect to spermatozoa and improved post thawed sperm quality (Pursel et al., 1978). It has been suggested that SDS enhances the cryoprotective properties of the egg yolk to protect the sperm membrane from

Three ejaculates from each boar are collected using gloved-hand method. During collection the semen is filtered through gauze and only sperm rich fractions are collected. Within 30 min after collection, semen volume, pH, sperm motility, concentration, percentage of live and dead sperm and morphology are determined. Only ejaculates with motility of ≥70% and

semen in pig (Crabo et al., 1971; Pursel et al., 1971).

insemination.

cryoinjuries (Buhr et al., 1996)

**4.1 Semen collection** 

**4. Boar semen cryopreservation methods** 

≥80 % morphologically normal are used for cryopreservation.

Fig. 1. Flow chart of the boar semen freezing processes, thawing and evaluation

Cryopreservation of Boar Spermatozoa: An Important Role of Antioxidants 145

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

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

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;

**A B**

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

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

**6. Capacitation like changes of the frozen-thawed boar semen and its** 

(Almlid and Johnson, 1988; Eriksson et al., 2001)

(Ball and Vo, 2001) and bull (Liu and Foote, 1998).

2005)(Fig. 2).

damage (B).

**influence on** *in vivo* **fertility** 

#### **4.2 Semen freezing and thawing procedures**

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.
