**3. Cryopreservation of animal spermatozoa**

Cryopreservation of boar semen need to be developed for AI in the pig industry due to a number of reasons including preservation of a good genetic resource, increase genetic improvement, distribution of genetic lines across countries and reduce boar transportation

Cryopreservation of Boar Spermatozoa: An Important Role of Antioxidants 143

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

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

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 insemination.

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 cryoinjuries (Buhr et al., 1996)

#### **4. Boar semen cryopreservation methods**
