Kampon Kaeoket

*Faculty of Veterinary Science, Semen laboratory, Department of Clinical Science and Public Health, Mahidol University Thailand* 

#### **1. Introduction**

Artificial insemination (AI) is one of the first reproductive biotechnologies has been established and developed in the pig production system. In most case, liquid stored semen or fresh semen is used for AI in commercial swine herds (Wagner and Thibier, 2000). The use of FT boar semen for AI is limited due to the low fertility outcomes compared to extended fresh semen (Johnson et al., 2000; Wagner and Thibier, 2000). The first success of boar semen cryopreservation was reported in 1956 (Polge, 1956) and the first pregnancy was achieved with FT boar semen using surgical insemination in 1970 (Polge, 1970). Currently, the attempt to develop the boar semen cryopreservation technique is ongoing. Nevertheless, the success of boar semen cryopreservation is relatively variable because the factors responsible for the cryosurvival of boar spermatozoa have not been entirely elucidated.

Cryopreservation of boar semen is useful for preservation of genetic resources, improve the genetic progress and enhance the transportation of genetic material across countries (Almlid and Hofmo, 1996; Johnson, 1998). In addition, the frozen-thawed (FT) boar semen is also used with other reproductive technologies, such as in vitro fertilization (IVF), embryo transfer (ET) and sex pre-selection (Gerrits et al., 2005). Unfortunately, the advancement of sperm cryopreservation in pigs is slow, partly due to the pig producer is satisfied with the liquid stored semen and low conception rate and litter size remain the major problems when using FT boar semen (Eriksson et al., 2002.). Under field conditions, low fertility is still obtained even using FT boar semen with a sufficient motility and number of spermatozoa for insemination (Johnson et al., 2000, Eriksson et al., 2002).

The use of frozen-thawed (FT) boar semen has been developed for artificial insemination (AI) in pig long time ago in Europe and USA (Larsson and Einarsson, 1976). In Thailand, few studies on boar semen cryopreservation have been established (Buranaamnuay et al., 2006 a,b). However, a great variation on the survival rate of post-thawed spermatozoa are obtained, due to the lack of biological background concerning the cryopreservation technique (Buranaamnuay et al., 2006 a,b). During the recent years, studies on FT boar semen have dramatically improved boar semen cryopreservation technique, for instance, optimum freezing protocols (Eriksson and Rodrigrez-Martinez, 2000), types of freezing package (Bwanga et al., 1991;Berger and Fisherleitner, 1992; Bwanga et al., 1991; Eriksson and Rodriguez-Martinez, 2000), semen centrifugation methods (Carvajal et al., 2004),

Cryopreservation of Boar Spermatozoa: An Important Role of Antioxidants 141

2005) in the semen extenders reduces the ROS formation and improve post-thawed sperm motility and viability of FT boar semen. In addition, it was found that the supplement of extended boar semen with 5 mM of cysteine improved the viability and functional status of

Nowadays, there are 2 techniques for cryopreservation in boar semen, traditional nitrogen method and controlled rate freezing method. Verheyen (1993) reported a significantly better post-thaw sperm outcome when computer controlled rate freezing was used compared to non-controlled rate freezing. In human, it has been reported that controlled rate freezer method provided significant superior post-thaw sperm motility, viability, and cryosurvival rate, compared with traditional nitrogen method (Petyim and Choavaratana, 2006). However, the study of Thalchil (1981) did not confirm the different outcome of these 2 methods. Besides breed-specific fertility, data from field trials found that the mean motility of frozen–thawed semen between Norwegian Landrace and Duroc boars was difference. In the different breeds of boars found the differences in membrane lipid composition, can neither explain the major differences in post-thaw survival and fertility between breeds

Major limitation of frozen-thawed semen (FT-boar semen) have been observed, i.e. low conception rate and low litter size after AI (Johnson et al., 2000; Buranaamnuay et al., 2006). The relatively low fertility of FT-boar is associated with many factors. It has been reported that reactive oxygen species (ROS) generation, induced by the cryopreservation process, can be responsible for mammalian sperm damage (Griveau and Le Lannou, 1997) ROS production has been associated with reduction of sperm motility and decreased capacity for sperm–oocyte fusion. Spermatozoa are sensitive to lipid peroxidation due to their high content of polyunsaturated fatty acids, and are unable of resynthesizing their membrane components, although this may not be the sole mechanism by which sperm function is impaired by ROS. Many studies have shown that the supplementation of antioxidants in extenders improved the qualities of both fresh boar semen (Bamba and Cran, 1992; Funahashi and Sano, 2005) and frozen boar semen (Breininger et al., 2005; Gadea et al., 2005; Pena et al., 2003; Roca et al., 2004; Roca et al., 2005). Earlier studies showed that the supplementation of some antioxidant such as, water soluble Vitamin E 200 µM to semen extenders for freezing boar spermatozoa reduced post-thaw ROS generation and improved sperm motility and viability (Pena et al., 2003). Funahashi and Sano (2005) reported that supplementation extended boar semen with glutathione or *L-*cysteine of 5 mM improved the viability and functional status of boar spermatozoa during liquid storage at 10 C for at least 14 day. A recent report (Gadea et al., 2005) demonstrated that supplementation with 1 mM of reduced glutathione to freezing media resulted in a protective effect on sperm function.

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

the chilled boar spermatozoa (Funahashi and Sano, 2005).

**3. Cryopreservation of animal spermatozoa** 

**2. History of cryopreservation** 

(Waterhouse et al., 2006).

thawing process (Eriksson and Rodrigrez-Martinez, 2000; Córdova-Izquierdo et al., 2006) and the supplement of some additives to the semen extender (Peña et al., 2003, Gadea et al., 2004; Roca et al., 2004, 2005).

Boar semen differs in several aspects from the semen of other domestic animals, for instance, the semen is produced in a large volume and highly sensitive to cold shock, the viability of the sperm cells is dramatically reduced when expose to temperatures below 15 C (Gilmore et al., 1996). Therefore, the manipulation of boar semen requires special consideration during cryopreservation process (Johnson et al., 2000). Many factors that should be concerned for the boar semen cryopreservation included composition of diluents, type and concentration of cryoprotective agent, equilibration time, cooling rate and thawing procedure.

The relatively low fertility of FT boar semen is associated with many factors including a highly sensitive plasma membrane of boar spermatozoa against the changing in temperature during cooling, freezing and thawing process (Holt, 2000; Watson, 2000). This problem is related to the lipid composition of the sperm plasma membrane. The plasma membrane of the boar spermatozoa contains a high level of polyunsaturated fatty acids (PUFAs) i.e., docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), and had a low cholesterol to phospholipids ratio. DPA and DHA are dominant fatty acids in the plasma membrane of boar spermatozoa (Johnson et al., 1969).

During cryopreservation, PUFAs decrease dramatically due to lipid peroxidation. This is initiated when the spermatozoa is attacked by reactive oxygen species (ROS) (De Lamirande and Gagnon, 1992; Sikka et al., 1995). In mammals, the major sources of ROS formation include leucocyte, defective and dead spermatozoa (Aitken et al., 1994; Silva, 2006). The excessive ROS formation influence sperm motility, mid-piece abnormalities and spermoocyte fusion (Chatterjee et al., 2001; Agarwal et al., 2005).

The supplement of antioxidant compounds and some fatty acid to the semen extender, to minimize ROS formation and protect the plasma membrane function, have been used in many species (Peña et al., 2003; Gadea et al., 2004; Roca et al., 2004; 2005; Maldjian et al., 2005). It has been demonstrated that the proportion of DHA was significantly higher in the semen diluted with an extender supplemented with n-3 enriched hen egg yolks compared with the semen diluted with normal hen egg yolks (Maldjian et al., 2005). However, no study has been demonstrated clearly whether or not the supplement of DHA could improve the quality of the boar spermatozoa after cryopresevation. Rooke et al. (2001) found that DHA supplement in the boar feed increase progressive motility and normal acrosome and decrease abnormal spermatozoa. Recently, Kaeoket et al. (2008) reported that the supplement of DHA-enriched fish oil improved the FT boar semen quality. It has been shown that the supplement of cryoprotective agents (e.g., glycerol and Equex®), cholesterol analogue (Zeng and Terada, 2001) and antioxidants (e.g., Vitamin E, alpha-tocopherol, glutathione, taurine, cysteine, butylated hydroxytoluene, superoxide dismutase and catalase) in the semen extenders does improve the freezing ability of spermatozoa of many species such as stallion (Aurich et al., 1997; Ball et al., 2001), bull (Beconi et al., 1993; Bilodeau et al., 2001), ram (Uysal and Bucak, 2007; Bucak et al., 2007), avian (Donoghue and Donoghue, 1997), boar (Cerolini et al., 2000) and some wildlife (Leibo and Songsasen, 2000). Studies have demonstrated that the supplement of alpha-tocopherol (Peña et al., 2003), butylated hydroxytoluene (Roca et al., 2004), superoxide dismutase and catalase (Roca et al.,
