Pathophysiology of lipid peroxidation

A variety of semen components, including morphologically abnormal spermatozoa, precursor germ cells and leucocytes are capable of generating ROS. Depending upon the nature, concentration, location and time of exposure, ROS may have beneficial or harmful effects (Agarwal and Saleh, Reference AGARWAL and SALEH2002). Under physiological conditions, spermatozoa produce small amounts of ROS such as superoxide anions, which effectively play a role in capacitation and acrosomal reactions (Agarwal et al., Reference AGARWAL, SALEH and BEDAIWY2003). Superoxide anions, hydroxyl radicals and hydrogen peroxide are some of the major ROS present in seminal plasma. The link between the poor quality of sperm and the increased generation of ROS lies in the formation of cytoplasmic droplets in abnormal spermatozoa (Agarwal and Saleh, Reference AGARWAL and SALEH2002; Agarwal et al., Reference AGARWAL, SALEH and BEDAIWY2003). During spermatogenesis, spermatozoa lose most of their cytoplasm while undergoing remarkable transformation of differentiation during their final stage of maturation. Under certain circumstances, the cytoplasm is transformed into cytoplasmic droplets. Such spermatozoa are thought to be immature and functionally defective and therefore capable of producing high concentrations of ROS (Sikka, Reference SIKKA2001; Agarwal et al., Reference AGARWAL, SALEH and BEDAIWY2003).

Limited production of ROS plays a physiological role in the regulation of sperm function. Recent studies have shown that incubation of spermatozoa with low levels of hydrogen peroxide (H₂O₂) stimulates sperm capacitation and the ability of spermatozoa to undergo the acrosome reaction and oocyte fusion (de Lamirande and Gagnon, Reference DE LAMIRANDE and GAGNON1995; Kefer et al., Reference KEFER, AGARWAL and SABANEGH2009). Likewise, nitric oxide was also found in the sperm capacitation and the acrosome reaction (Zini et al., Reference ZINI, DE LAMIRANDE and GAGNON1996). Sperm capacitation is a fundamental step which prepares spermatozoa to reach the site of fertilisation at the appropriate time and rapidly undergo the acrosome reaction upon contacting the zona pellucida of the ovum. An increased formation of ROS has been correlated with a reduction in sperm motility (Agarwal et al., Reference AGARWAL, IKEMOTO and LOUGHLIN1994). The link between ROS and reduced sperm motility has been associated with the cascade of events that result in decrease in axonemal protein phosphorylation and sperm immobilisation (de Lamirande and Gagnon, Reference DE LAMIRANDE and GAGNON1995). These alterations can cause a decrease in the antioxidant capacity of the spermatozoa, which ultimately leads to the peroxidation of membrane phospholipids (Griveau et al., Reference GRIVEAU, DUMONT, RENARD, CALLEGARI and LE LANNOU1995).

One of the very important sources of lipid peroxidation is the mitochondria, which plays a major role in the production of ROS through the nicotinamide adenine dinucleotide (NADH)-dependent oxido-reductase system (Hallak et al., Reference HALLAK, SHARMA, PASQUALOTTO, RANGANATHAN, THOMAS and AGARWAL2001). Mitochondria are present in sufficient amounts in gametes which provide mechanical energy for motility. The inner mitochondrial membrane potential (MMP) is very important in regulating sperm function. Wang et al. (Reference WANG, SHARMA, GUPTA, GEORGE, THOMAS and FALCONE2003) concluded that MMP decreased in spermatozoa of infertile subjects with high levels of ROS production and have a positive correlation with sperm concentration. Mitochondrion also plays a key role in the process of apoptosis. High levels of ROS disrupts the outer and inner mitochondrial membrane which results in the release of cytochrome C protein and activate caspases to induce apoptosis (Wang et al., Reference WANG, SHARMA, GUPTA, GEORGE, THOMAS and FALCONE2003). Elevated levels of cytochrome C in seminal plasma indicates significant mitochondrial damage by ROS.

DNA damage and cross linking proteins can also deteriorate semen quality (Sharma and Agarwal, Reference SHARMA and AGARWAL1996). Exposure of sperm to artificially produced ROS causes DNA damage in the form of modification of all bases, frame shifts, DNA cross-links and chromosomal arrangements (Duru et al., Reference DURU, MORESHEDI and OEHNINGER2000). Spermatozoa with dysfunctional DNA are unable to fertilize the oocyte. Therefore, fertilisation rate decreases with increasing DNA damage (Sun et al., Reference SUN, JURISICOVA and CASPER1997).

Ameliorative role of antioxidants

Antioxidants, in general, are compounds and reactions that dispose, scavenge and suppress the formation of ROS, or oppose their action. Studies have shown that antioxidants have widespread effects on male fertility. These protect spermatozoa from ROS producing abnormal spermatozoa, scavenges ROS produced by leucocytes, prevents DNA fragmentation, reduce cryodamage to spermatozoa and blocks premature sperm maturation (Makker et al., Reference MAKKER, AGARWAL and SHARMA2009). Interestingly, the seminal plasma has been endowed with an array of enzymatic and non-enzymatic antioxidants which protect the sperm integrity against the negative impacts of ROS. These include endogenous antioxidants which comprise those compounds present in seminal plasma and spermatozoa. Seminal plasma contains four main types of enzymatic antioxidants; superoxide dismustase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GRD), in addition to a wide range of non-enzymatic antioxidants like ascorbate, urate, vitamin E, pyruvate, glutathione, albumin, vitamin A, ubiquitol, taurine and hypotaurine (Makker et al., Reference MAKKER, AGARWAL and SHARMA2009). Another group of seminal antioxidants include metal-binding proteins such as albumin, ceruloplasmin, metallothionein, transferrin, ferritin, and myoglobin (Sanocka and Kurpisz, Reference SANOCKA and KURPISZ2004; Makker et al., Reference MAKKER, AGARWAL and SHARMA2009). Physiologically, the above mentioned seminal antioxidants are sufficient to neutralise the production of ROS, however under certain conditions the endogenous antioxidant's capacity is limited. The neutralizing machinery depends upon exogenous sources to stabilise the equilibrium between ROS and antioxidants.

Antioxidative mechanism of vitamin E

Vitamin E is a major chain-breaking antioxidant in the sperm membrane. It scavenges all three types of free radicals, namely, superoxide, H₂O_2, and hydroxyl radical (Makker et al., Reference MAKKER, AGARWAL and SHARMA2009). Zaniboni et al. (Reference ZANIBONI, RIZZI and CEROLINI2006) reported that vitamin E inhibits lipid peroxidation in biological membranes acting as a scavenger of lipid peroxyl [LOO^-] and alkoxyl [LO^-] radicals. Vertuani et al. (Reference VERTUANI, ANGUSTI and MANFREDINI2004) concluded that vitamin E acts using at least two different mechanisms. Firstly, it directly scavenges ROS. Secondly, this vitamin up-regulates antioxidant enzymes such as SOD, GPX, GRD, CAT and NADPH (Nicotinamide adenine dinucleotide phosphate). This vitamin also acts synergistically with selenium to energize the enzymatic role of GPX (Surai et al., Reference SURAI, KOSTJUK, WISHART, PHERSON, SPEAKE, NOBLE, IONOV and KUTZ1998). Additionally, this vitamin has been known to reduce Fe⁺²-induced lipid peroxidation (Surai et al., Reference SURAI, BRILLARD, SPEAKE, BLESBOIS, SEIGNEURIN and SPARKS2000; Zaniboni et al., Reference ZANIBONI, RIZZI and CEROLINI2006). According to Eid et al. (Reference EID, EBEID and YOUNIS2006), vitamin E is naturally present in the sperm membrane and seminal plasma and it can protect the sperm in two ways. Firstly, during spermatogenesis in the testes, vitamin E protects the biological membrane from lipid peroxidation. Secondly, vitamin E reduces lipid peroxidation in seminal plasma and maintains an adequate viability of sperm to complete the fertilisation process. Finally, dietary vitamin E supplementation enhances sperm number and quality.

Vitamin E also indirectly acts on other components of semen to stabilise the sperm. For example, vitamin E decreases semen homocysteine which is a defective amino acid formed from demethylation of methionine. Homocysteine has been established as an independent risk factor for coronary heart diseases (Sonmez et al., Reference SONMEZ, YUCE, TURK, SONMEZA, YUCE and TURK2007). Wallock et al. (Reference WALLOCK, AMURA, MAYR, JOHNSTON, AMES and JACOB2001) reported that an increase in homocysteine is positively correlated with a reduction of semen parameters. The increased concentration of homocysteine blocks intracellular protein-carboxyl methylation reactions which results in the inhibition of sperm motility. Sonmez et al. (Reference SONMEZ, YUCE, TURK, SONMEZA, YUCE and TURK2007) found that homocysteine levels decreased and the progressive sperm motility significantly increased in the simultaneous homocysteine and vitamin E treated animals and concluded that a decrease in homocysteine may be explained by the prevention of peroxidative damage by free radicals in response to vitamin E treatment. The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are known as an index of sperm membrane stabilizing capacity (Yousef et al., Reference YOUSEF, ABDALLAH and KAMEL2003). The release of transaminase into the seminal plasma is also a useful indicator of injury to chicken spermatozoa. When the sperm cell membrane is damaged, AST and ALT are released into the extracellular medium. Studies have shown that AST and ALT decreased in response to vitamin E supplementation and semen characteristics improved, indicating an antioxidative role of this vitamin (Al-Daraji et al., Reference AL-DARAJI, AL-ATHARI and AL-MASHADANI2000; Yousef et al., Reference YOUSEF, ABDALLAH and KAMEL2003; Biswas et al., 2009).

A variety of defence mechanisms comprising antioxidant enzymes, biomolecules and vitamins are available which help to reduce the risk of ROS and appear to play vital roles in the survival and functioning of spermatozoa. In the last few decades, the role of exogenous vitamin E as an antioxidant in poultry diets for the improvement of avian male fertility has been immensely practised. Therefore, the aim of present document is to review the previous available literature on avian semen quality as affected by supplementation of dietary vitamin E.

Effect of vitamin E supplementation on avian semen quality

Vitamin E has traditionally been known as an anti-sterility vitamin. Vitamin E is the most important lipid antioxidant. This vitamin was first detected in turkey semen in 1981 and later also confirmed in chicken semen. Now the role of vitamin E as an antioxidant in poultry feed is well recognised. The importance of this vitamin can be seen from the fact that it constitutes 88% of chicken semen antioxidant capacity. Structurally, vitamin E has been categorised as α (alpha), β (beta) and γ (gamma) tocopherol. α-tocopherol is considered to be the major form of vitamin E in spermatozoa, followed by γ- and β-tocopherol (Surai et al., Reference SURAI, KUTZ, WISHART, NOBLE and SPEAKE1997). Vitamin E concentration in chicken semen is about 0.46 µg/ml without supplementation and this amount was shown to increase to 1.04 µg/ml when this vitamin was included at the level of 200 mg/kg in the diet of chickens (Surai et al., Reference SURAI, KUTZ, WISHART, NOBLE and SPEAKE1997).

To enhance avian male reproductive performance, dietary supplementation of vitamin E is used routinely in poultry production. The results obtained may vary depending upon the dose and duration of vitamin E supplementation, genetics, age and measurement criteria (Siegel et al., Reference SIEGEL, PRICE, MELDRUM, PICARD and GERAERT2001). The dietary increase in the Vitamin E inside semen was found to result in a significant reduction in the susceptibility to lipid peroxidation (Niki, Reference NIKI1993; Lin et al., Reference LIN, CHANG, YANG, LEE and HSU2005). Biswas et al. (Reference BISWAS, MOHAN, SASTRY and TYAGI2007) demonstrated in Japanese quail (Coturnix coturnix japonica) that supplemental vitamin E at the rate of 150 IU/kg of feed improved foam discharge and cloacal gland size. Biswas et al. (Reference BISWAS, MOHAN, SASTRY and TYAGI2007) also found that fertility was lower in the absence of vitamin E in the basal diet and when vitamin E was provided, fertility was restored in the experimental quails. Marzoni et al. (Reference MARZONI, CABRAS, GUERZILOV and ROMBOLI2000) concluded from his study in open air reared pheasants that a diet supplemented with vitamin E is an effective means to improve their fertilizing ability.

Unlike vitamin C, this vitamin cannot be synthesised by the body. Therefore, vitamin E is an important constituent of every feed formulation. Nutritionist have recommended that poultry feed should contain at least 10 mg vitamin E/kg of feed for optimum reproductive performance and long term omission of this vitamin may result in negative consequences (Whitehead and Portsmouth, Reference WHITEHEAD and PORTSMOUTH1989). Lin et al. (Reference LIN, CHANG, YANG, LEE and HSU2005) demonstrated in 39 week old Taiwan native cockerels that supplemental vitamin E at the rate of 40 and 160 mg/kg plays a very significant role in improving semen traits, particularly sperm liveability and motility, however, sperm concentration and plasma testosterone concentration improved only when feed was supplemented with 80 or 160 mg/kg of vitamin E. Cereloni et al. (2006) found that sperm concentration was significantly improved in male broiler breeders by the dietary addition of vitamin E at the rate of 200 mg/kg. Biswas et al. (2009) concluded that addition of vitamin E (200mg) in the basal diet of Kadaknath cockerels, an Indian local breed, famous for poor production, significantly decreased the proportion of abnormal and dead spermatozoa and therefore improved fertility. In another experiment, Cereloni et al. (2006) concluded that semen volume, liveability, total sperm concentration and motility were significantly increased by the inclusion of vitamin E at the rate of 300 mg/kg in broiler breeder diets.

It has been documented that fertility in male birds decreased with increasing age (Hocking and Bernard, Reference HOCKING and BERNARD2000; Romero-Sanchez et al., Reference ROMERO-SANCHEZ, PLUMSTEAD, LEKSRISOMPONG, BRANNAN and BRAKE2008). Age related decreases in fertility in cockerels has been linked with low levels of vitamin E in the testes and this decline can be restored with vitamin E supplementation up to 200 mg/kg (Surai et al., Reference SURAI, KUTZ, WISHART, NOBLE and SPEAKE1997; Reference SURAI, BRILLARD, SPEAKE, BLESBOIS, SEIGNEURIN and SPARKS2000). In addition, supplementation of selenium (Se) enhances the role of vitamin E by increasing the activity of GSH, reducing the susceptibility of spermatozoa to lipid peroxidation (Surai et al., Reference SURAI, KOSTJUK, WISHART, PHERSON, SPEAKE, NOBLE, IONOV and KUTZ1998). Eid et al. (Reference EID, EBEID and YOUNIS2006) reported that vitamin E protects sperms from damage caused by oxidative stress, which is translated into enhanced sperm motility, sperm count, reduced numbers of dead sperm and a decline in TBARS (thiobarbituric acid reactive substances). Biswas et al. (2009) found that supplementation of 150 IU/kg vitamin E caused a decrease in the percentage of dead sperm and abnormal spermatozoa. One can therefore argue that this beneficial effect under oxidative stress is due to the antioxidant properties of vitamin E.

In vitro studies have also shown that vitamin E is a major chain-breaking antioxidant in the sperm membrane. Cryopreservation and thawing techniques are associated with a significant reduction in sperm liveability and motility induced by ROS and these effects can be effectively combated by the addition of vitamin E to cryoprotectants (Surai et al., Reference SURAI, BRILLARD, SPEAKE, BLESBOIS, SEIGNEURIN and SPARKS2000). In vitro studies have demonstrated the inhibition of lipid peroxidation by the addition of vitamin E into semen diluents. Donoghue and Donoghue (Reference DONOGHUE and DONOGHUE1997) demonstrated that a moderate concentration of 80 µg/ml vitamin E tends to increase the fertilizing potential of turkey semen stored at 24°C for 24 hours. In the same experiment, the addition of vitamin E to the diluted semen resulted in improved membrane integrity, better sperm survivability along with higher motility and lower lipid peroxidation. In vitro studies, Surai et al. (Reference SURAI, KOSTJUK, WISHART, PHERSON, SPEAKE, NOBLE, IONOV and KUTZ1998) also demonstrated that the addition of vitamin E decreased oxidative stress significantly in teste homogenates.