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Comparative Effect Of Alpha Lipoic Acid And Butylated Hydroxytoulene On Post Thaw Quality Of Buck Semen

By: Muhammad Khurram Shahzad | Prof. Dr. Mian Abdul Sattar.
Contributor(s): Dr. Mushtaq Ahmad | Dr. Muhammad Yasin Tipu.
Material type: materialTypeLabelBookPublisher: 2015Description: 40p.Subject(s): Department of TheriogenologyDDC classification: 2254-T Dissertation note: Amongst different livestock species, goats and sheep are the major source of livelihood for over a million livestock farmers in Pakistan. Total goat population in Pakistan is estimated to be 66.6 million. These animals are mostly kept by small holders for whom these are only source of their livelihood. Milk production from goats is 0.822 million tonnes while mutton production from both sheep and goats is 0.657 million tonnes (Anonymous 2014). Pakistani people mostly prefer the goat meat over sheep. All irrigated areas of Punjab including district Faisalabad, Sahiwal, Sargodha, Jhang, Jhelum, Lahore and Multan are the habitat of Makhi Cheeni Beetal goats. The color of its body coat is red spotted or golden brown with white patches. Its body is very well developed and compact. Males have long spiraled horns while females have shorter. It has roman nose with pendulous broad and long ears. It has long teats and well developed udder. Female weighs 37kg and males 46kg. Twins or triplets births are more than 50%. In 130 days of lactation period, there is 290 liters milk yield (Shah et al. 2001). Some breeds of goats especially dairy goats have more demand than the others and these bucks are not available everywhere. To cope with this situation artificial insemination techniques is necessary. Artificial insemination plays a great role in increasing the economics by spreading the superior genetics within a short period of time. Semen is processed by different methods but cryopreservation is considered to be the best method. Cryopreservation has been reported to compromise the quality of processed semen resulting in the loss of sperm motility, viability, in-vivo fertilizing ability, deterioration of plasma membrane and acrosomal integrity, apoptosis and damage of deoxyribonucleic acid (DNA) (Medeiros et al. 2002; Purdy 2006a). Sperm damage may occur due to various factors like osmotic stress, oxidative stress, low-temperature exposure and combination of different factors (Sarıözkan et al. 2009). Thawing of semen may also cause osmotic changes and the sperm quality is further decreased. It is generally accepted that sperm viability is reduced by as much as 50% during the process of semen cryopreservation (Watson, 2000). Extension of buck semen with egg yolk containing extender may be more injurious to sperms. This is due to the presence of coagulating enzymes of bulbourethral origin named as egg yolk coagulating enzymes (EYCE). EYCE decreases the tenacity of chilled or frozen semen (Roy, 1957). EYCE also catalyze the conversion of egg yolk lecithin into lsolecithin and fatty acid, thus sperm membrane become more fusogenic due to hydrolysis. So there is increase in chromatin decondensation and acrosomal reaction that is harmful for sperm (Leboeuf et al. 2000). Due to excess of poly unsaturated fatty acids (PUFA) in sperms, they are more susceptible to lipid peroxidation (Cassani et al. 2005). Lipid peroxidation of PUFA lead to production of reactive oxygen species (ROS) (Alvarez et al. 1995). Small amount of ROS are normally involved in capacitation, acrosmal reaction and ultimately fertilizing ability of sperms. But when the ROS are produced in excess Introduction 3 amount, these may compromise the enzymatic function and sperm fertility (Baumber et al. 2000). At 4-5 ºC the motility and plasma membrane integrity is decrease with the passage of time which ultimately leads to decrease in fertility. One of the cause of this decrease is production of ROS by the lipid peroxidation of spermatozoa’s membrane (Storey et al. 1998). Major decrease in sperm motility and fertility occur during phase transition from liquid crystalline to gel phase (Chakrabarty et al. 2007). Lipid peroxidation leads to irreversible loss in motility and damage to DNA of sperm (Maxwell et al. 1996). Motility of sperm is adversely effected with ROS, when the ROS harm the plasma membrane and acrosomal integrity which ultimately leads to fragmentation of DNA. Sperms have their own antioxidants system which include the glutathione (GSH) , GSH peroxides, superoxide dismutase, catalase and chelators of transferrin, lactoferrin and ceruplasmin (Agarwal et al. 2002). Normally the ROS production and scavenging are in equilibrium but during the semen preservation the excessive production of ROS (superoxide, hydroxyl, hydrogen peroxide, nitric oxide, peroxynitrile) with low level of scavenging system and antioxidants leads to oxidative stress. During the process of freezing and thawing the natural antioxidants systems are unable to stop lipid peroxidation. Therefore a powerful antioxidant system should be used to avoid the cryo-injuries and lipid peroxidation (Irvine 1996). Different antioxidants are being used i.e. fetuin (F), amino acid (AS), cysteine (CY) taurine, glutathione (GSH) glutathione peroxidase (GSH-PX), catalase (CAT), superoxide dismutase (SOD) glutamine, hyaluronan, trehalose, Alpha lipoic acid (ALA) and Butylated Hydroxytoulene (BHT) (Atessahin et al. 2008; Bucak et al. 2009; Taşdemir et al. 2014). Addition of antioxidants to semen extenders are considered to improve the quality of semen (Rao et al. 2013). ALA is a short chain fatty acid which act as an antioxidant in both aqueous and lipid environments, its therapeutic effects in other tissues like brain (Piotrowski et al. 2001), heart, kidneys and testicles has already been Introduction 4 discussed. It is called as universal antioxidant because of its effect in different parts of body. It is not only involve in scavenging the ROS but also activate the body antioxidants systems against ROS. ALA reduced to dithiol form called dihydrolipoic acid (DHLA) which is an excellent antioxidant (Handelman et al. 1994). ALA also regenerates vitamin C from reduced vitamin C in the presence of glutathione (GSH) which also enhance the antioxidant activity (Ibrahim et al. 2008). BHT, a phenolic lipophilic antioxidant that has antiviral activity, have the ability to relieve the cold shock in spermatozoa from several animal species. It stops the auto oxidation by converting the peroxide radical to hydroperoxide as it is also called as synthetic analogues of Vit E (Memon et al. 2011). BHT acts as a membrane lipid protectant which reduces the changes in permeability of sperm plasma membrane in cold shock (Graham et al. 1992). BHT minimizes the effect of cold shock on semen (Shoae et al. 2008), boar (Roca et al. 2004) and goat (Khalifa et al. 2008).
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Amongst different livestock species, goats and sheep are the major source of livelihood for over a million livestock farmers in Pakistan. Total goat population in Pakistan is estimated to be 66.6 million. These animals are mostly kept by small holders for whom these are only source of their livelihood. Milk production from goats is 0.822 million tonnes while mutton production from both sheep and goats is 0.657 million tonnes (Anonymous 2014). Pakistani people mostly prefer the goat meat over sheep.
All irrigated areas of Punjab including district Faisalabad, Sahiwal, Sargodha, Jhang, Jhelum, Lahore and Multan are the habitat of Makhi Cheeni Beetal goats. The color of its body coat is red spotted or golden brown with white patches. Its body is very well developed and compact. Males have long spiraled horns while females have shorter. It has roman nose with pendulous broad and long ears. It has long teats and well developed udder. Female weighs 37kg and males 46kg. Twins or triplets births are more than 50%. In 130 days of lactation period, there is 290 liters milk yield (Shah et al. 2001).
Some breeds of goats especially dairy goats have more demand than the others and these bucks are not available everywhere. To cope with this situation artificial insemination techniques is necessary. Artificial insemination plays a great role in increasing the economics by spreading the superior genetics within a short period of time.
Semen is processed by different methods but cryopreservation is considered to be the best method. Cryopreservation has been reported to compromise the quality of processed semen resulting in the loss of sperm motility, viability, in-vivo fertilizing ability, deterioration of plasma membrane and acrosomal integrity, apoptosis and damage of deoxyribonucleic acid (DNA) (Medeiros et al. 2002; Purdy 2006a). Sperm damage may occur due to various factors like osmotic stress, oxidative stress, low-temperature exposure and combination of different factors (Sarıözkan et al. 2009). Thawing of semen may also cause osmotic changes and the sperm quality is further decreased. It is generally accepted that sperm viability is reduced by as much as 50% during the process of semen cryopreservation (Watson, 2000).
Extension of buck semen with egg yolk containing extender may be more injurious to sperms. This is due to the presence of coagulating enzymes of bulbourethral origin named as egg yolk coagulating enzymes (EYCE). EYCE decreases the tenacity of chilled or frozen semen (Roy, 1957). EYCE also catalyze the conversion of egg yolk lecithin into lsolecithin and fatty acid, thus sperm membrane become more fusogenic due to hydrolysis. So there is increase in chromatin decondensation and acrosomal reaction that is harmful for sperm (Leboeuf et al. 2000). Due to excess of poly unsaturated fatty acids (PUFA) in sperms, they are more susceptible to lipid peroxidation (Cassani et al. 2005). Lipid peroxidation of PUFA lead to production of reactive oxygen species (ROS) (Alvarez et al. 1995). Small amount of ROS are normally involved in capacitation, acrosmal reaction and ultimately fertilizing ability of sperms. But when the ROS are produced in excess
Introduction
3
amount, these may compromise the enzymatic function and sperm fertility (Baumber et al. 2000). At 4-5 ºC the motility and plasma membrane integrity is decrease with the passage of time which ultimately leads to decrease in fertility. One of the cause of this decrease is production of ROS by the lipid peroxidation of spermatozoa’s membrane (Storey et al. 1998). Major decrease in sperm motility and fertility occur during phase transition from liquid crystalline to gel phase (Chakrabarty et al. 2007). Lipid peroxidation leads to irreversible loss in motility and damage to DNA of sperm (Maxwell et al. 1996). Motility of sperm is adversely effected with ROS, when the ROS harm the plasma membrane and acrosomal integrity which ultimately leads to fragmentation of DNA. Sperms have their own antioxidants system which include the glutathione (GSH) , GSH peroxides, superoxide dismutase, catalase and chelators of transferrin, lactoferrin and ceruplasmin (Agarwal et al. 2002). Normally the ROS production and scavenging are in equilibrium but during the semen preservation the excessive production of ROS (superoxide, hydroxyl, hydrogen peroxide, nitric oxide, peroxynitrile) with low level of scavenging system and antioxidants leads to oxidative stress. During the process of freezing and thawing the natural antioxidants systems are unable to stop lipid peroxidation. Therefore a powerful antioxidant system should be used to avoid the cryo-injuries and lipid peroxidation (Irvine 1996).
Different antioxidants are being used i.e. fetuin (F), amino acid (AS), cysteine (CY) taurine, glutathione (GSH) glutathione peroxidase (GSH-PX), catalase (CAT), superoxide dismutase (SOD) glutamine, hyaluronan, trehalose, Alpha lipoic acid (ALA) and Butylated Hydroxytoulene (BHT) (Atessahin et al. 2008; Bucak et al. 2009; Taşdemir et al. 2014). Addition of antioxidants to semen extenders are considered to improve the quality of semen (Rao et al. 2013). ALA is a short chain fatty acid which act as an antioxidant in both aqueous and lipid environments, its therapeutic effects in other tissues like brain (Piotrowski et al. 2001), heart, kidneys and testicles has already been
Introduction
4
discussed. It is called as universal antioxidant because of its effect in different parts of body. It is not only involve in scavenging the ROS but also activate the body antioxidants systems against ROS. ALA reduced to dithiol form called dihydrolipoic acid (DHLA) which is an excellent antioxidant (Handelman et al. 1994). ALA also regenerates vitamin C from reduced vitamin C in the presence of glutathione (GSH) which also enhance the antioxidant activity (Ibrahim et al. 2008). BHT, a phenolic lipophilic antioxidant that has antiviral activity, have the ability to relieve the cold shock in spermatozoa from several animal species. It stops the auto oxidation by converting the peroxide radical to hydroperoxide as it is also called as synthetic analogues of Vit E (Memon et al. 2011). BHT acts as a membrane lipid protectant which reduces the changes in permeability of sperm plasma membrane in cold shock (Graham et al. 1992). BHT minimizes the effect of cold shock on semen (Shoae et al. 2008), boar (Roca et al. 2004) and goat (Khalifa et al. 2008).

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