**3. Effect of Reactive Oxygen Species (ROS)**

Boar sperm are highly sensitive to peroxidative damage due to the high content of unsatu‐ rated fatty acids in the phospholipids of the sperm plasma membrane [3,4] and the correla‐ tion of low antioxidant capacity of boar seminal plasma and lipid-peroxidation [5]. It has been reported in sperm freezing of human [6], bull [7] and mouse [8] that is associated with ROS level and oxidative stress. Moreover, the process of freezing and thawing bovine sper‐ matozoa can generate the ROS [9], DNA damage [10], cytoskeleton alterations [11], inhibi‐ tion of the sperm–oocyte fusion [12] and can affect the sperm axoneme that is influenced on the sperm motility [13].

The lipid-peroxidation of membrane phospholipid bound docosahexaenoic acid (DHA) has been presented as one of the major factors that limit the sperm motility in vitro. Semen sam‐ ples show high sperm variability in lifespan and, consequently, in susceptibility toward lip‐ id peroxidation. Therefore, it is postulated that there is also cell-to-cell variability in DHA content in human spermatozoa and that the content of the main substrate of lipid peroxida‐ tion (DHA) is critical and highly regulated during the sperm maturation process. Several studies have been performed to analyze the fatty acid content of germ cells and sperm at different stages of maturation, including in vivo studies in animal models, and in vitro ap‐ proaches in human spermatozoa. One of the consequences of defective sperm maturation in the seminiferous epithelium is the retention of residual cytoplasm. This residual cytoplasm, which is attached to the midpiece and retronuclear area of the sperm head, has been shown to produce high levels of reactive oxygen species (ROS) [14-16]. In addition, the membranes enclosing the residual cytoplasm are enriched in polyunsaturated fatty acids such as DHA [17,18]. The combination of high polyunsaturated fatty acid content and high ROS produc‐ tion in these immature sperm has been shown to lead to increased lipid peroxidation and subsequent loss of sperm function [14,15]. ROS-mediated damage to human spermatozoa was characterized in the early 1980s [19-24] and has been shown by many authors to be an important factor in the pathogenesis of male infertility [14,25-27].

**2. Feed supplement to increase boar semen quality**

18 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

**3. Effect of Reactive Oxygen Species (ROS)**

the sperm motility [13].

The semen quality depends on individual, breed, season, confinement and boar health. It was found that the dietary supplements of antioxidants, vitamins and/or minerals can in‐ crease libido and semen characteristics in boars. Additions of antioxidants in seminal plas‐ ma or semen extender play an important role on boar semen storability. Semen with a normal motility contains higher polyunsaturated fatty acids (PUFAs) in cell membrane has that that having a low motility [1]. Short life span spermatozoa usually presented in low an‐ tioxidant condition resulting from the high lipid peroxidation of sperm plasma membrane. Spermatozoa in low antioxidants of seminal plasma also show a lower sperm motility, via‐ bility and normal morphology than spermatozoa in normal seminal plasma (Table 1) [1,2]. The feed supplements were expected to improve the semen quality by increasing the num‐ ber of sperm per ejaculation, motility, viability and antioxidant in cell and seminal plasma. However, it depends on the initial performance of the boar influencing on successfully im‐ proving semen quality. Therefore, the key roles of feed supplement containing the rich of PUFAs, vitamins and minerals to improve the semen quality are increasing the antioxidant to reduce the plasma membrane damages from ROS and increase the amount of PUFAs in sperm plasma membrane that may increase the percentage of sperm motility and vitality.

Boar sperm are highly sensitive to peroxidative damage due to the high content of unsatu‐ rated fatty acids in the phospholipids of the sperm plasma membrane [3,4] and the correla‐ tion of low antioxidant capacity of boar seminal plasma and lipid-peroxidation [5]. It has been reported in sperm freezing of human [6], bull [7] and mouse [8] that is associated with ROS level and oxidative stress. Moreover, the process of freezing and thawing bovine sper‐ matozoa can generate the ROS [9], DNA damage [10], cytoskeleton alterations [11], inhibi‐ tion of the sperm–oocyte fusion [12] and can affect the sperm axoneme that is influenced on

The lipid-peroxidation of membrane phospholipid bound docosahexaenoic acid (DHA) has been presented as one of the major factors that limit the sperm motility in vitro. Semen sam‐ ples show high sperm variability in lifespan and, consequently, in susceptibility toward lip‐ id peroxidation. Therefore, it is postulated that there is also cell-to-cell variability in DHA content in human spermatozoa and that the content of the main substrate of lipid peroxida‐ tion (DHA) is critical and highly regulated during the sperm maturation process. Several studies have been performed to analyze the fatty acid content of germ cells and sperm at different stages of maturation, including in vivo studies in animal models, and in vitro ap‐ proaches in human spermatozoa. One of the consequences of defective sperm maturation in the seminiferous epithelium is the retention of residual cytoplasm. This residual cytoplasm, which is attached to the midpiece and retronuclear area of the sperm head, has been shown to produce high levels of reactive oxygen species (ROS) [14-16]. In addition, the membranes

To a first approximation, the process of lipid peroxidation involves the initial abstraction of a hydrogen atom from the bis-allylic methylene groups of polyunsaturated fatty acids, mainly DHA, by molecular oxygen. This leads to molecular rearrangement to a conjugated diene and addition of oxygen, resulting in the production of lipid peroxide radical. This per‐ oxyradical can now abstract a new hydrogen atom from an adjacent DHA molecule leading to a chain reaction that ultimately results in lipid fragmentation and the production of malo‐ naldehyde and toxic shortchain alkanes (e.g., propane). These propagation reactions are mediated by oxygen radicals. DHA is the major polyunsaturated fatty acid in sperm from a number of mammalian species, including the human, accounting in this species for up to 30% of phospholipid-bound fatty acid and up to 73% of polyunsaturated fatty acids. At the same time, DHA is the main substrate of lipid peroxidation, accounting for 90% of the over‐ all rate of lipid peroxidation in human spermatozoa [23,28].


**Table 1.** Semen characteristics and antioxidant capacity in seminal plasma of boars having normal and low sperm motility (means ± SD)

Lipid peroxidation has profound consequences in biological membranes. The generation of the polar lipid peroxides ultimately results in the disruption of the membrane hydrophobic packing, inactivation of glycolytic enzymes, damage of axonemal proteins (loss of motility), acrosomal membrane damage, and DNA alterations [29,30]. Oxidation of phospholipidbound DHA has been shown to be the major factor that determines the motile lifespan of sperm in vitro [6,31,32]. Three basic factors determine the overall rate of lipid peroxidation of sperm in vitro: oxygen concentration and temperature in the medium (OXIDANT), the presence of antioxidant defenses (ANTIOXIDANT), and the content of membrane-bound DHA (SUBSTRATE). Thus, the higher the temperature and the concentration of oxygen in solution, the higher the rate of lipid peroxidation as measured by malonaldehyde produc‐ tion [24]. In boar, total antioxidant in seminal plasma relates to percentage of normal sperm morphology and plasma membrane. The low storability semen has presented the high plas‐ ma membrane damage from ROS, which was resulted from low amount of antioxidant in seminal plasma [2]. Moreover, the semen which having poor normal sperm morphology has shown the low level of antioxidant in seminal plasma (Table 1) [1].

optimal fertility in pig spermatozoa, as being in human sperm [28,36,37], then it is possible that supplement 22:6(n-3) PUFAs on boar diets to improve the spermatogenesis. This sup‐ plementation may increase from either a deficit of (n-3) fatty acids or an increasing synthesis of 22:6(n-3) from 18:3(n-3) to competition between (n-6) and (n-3) fatty acids [38]. The tuna oil supplementing on the boar diet can increase the percentages of sperm cells with progres‐ sive motility, the proportion of live sperm, normal acrosome head, and normal morphology [39]. It was found that boars fed withcommercially available product containing DHA, vita‐ min E and selenium (PROSPERM®, Minitube America, Inc., Minneapolis, MN) for 16 weeks had a higher sperm concentration, number of sperm/ejaculate, and sperm motility compar‐ ing with control group [40]. In many experiments, 8-week period was used as the control period because spermatogenesis in boars requires 34–39 d and epididymal transport in‐ volves another 9–12 d [41]. It is not surprising that a 7–8 week period may be necessary after

Improvement of Semen Quality by Feed Supplement and Semen Cryopreservation in Swine

http://dx.doi.org/10.5772/51737

21

The research on semen cryopreservation in boar is limited even though the procedures have been studied during the past 60 years [43-47]. The advantages for development of frozen se‐ men include the preservation of the good genetic resource, the distribution of superior ge‐ netic boars, and the improvement of the transportation of sperm across countries [48]. However, the utilization of frozen-thawed (FT) semen prepared for artificial insemination (AI) at present is estimated to be less than 1% of all insemination worldwide. The most im‐ portant reasons are the poor sperm quality after cryopreservation and a lower fertilizing ca‐ pacity of FT semen, when used for conventional AI compared to fresh semen. Poor sperm quality frequently found in FT boar semen is partly due to a high sensitivity of the boar sperm to rapid cooling to a few degrees above 0C, the so-called "cold shock", which the sperm have to traverse during cryopreservation process. This is evidenced by the loss of via‐ ble sperm and by more capacitation-like changes in the viable sperm [49]. These changes re‐ sult in a shorter survival time of the FT sperm in the female genital tract in comparison to its

**7. Factors affecting the success of boar semen cryopreservation**

packages, and the method of freezing and thawing of the semen, for example [48].

Boar semen differs in several respects from the semen of other domestic animals. It is pro‐ duced in large volume (200 to 250 ml) and is extremely sensitive to cold shock. The success of freezing boar semen depends on both internal and external factors. Internal factors in‐ clude the inherent characteristics of sperm and the existing differences among boars and ejaculates, while external factors are composed of the composition of the extenders, freezing

dietary supplementation [40,42].

**6. Boar semen cryopreservation**

fresh and liquid-preserved counterparts [50,51].

The balance between these key factors determines the overall rate of peroxidation in vitro. In this system, the substrate seems to play a key role. The main substrates for lipid peroxida‐ tion are polyunsaturated fatty acids, especially docosahexaenoic acid.
