Effect Of Cholesterol-Loaded Cyclodextrin (Clc) Addition On Egg Yolk Ratio In Semen Extender And Post-Thaw Quality Of Buffalo Bull Sperm
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Publisher: 2014 Dissertation note: Abstract
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Comparative Effect Of Alpha Lipoic Acid And Butylated Hydroxytoulene On Post Thaw Quality Of Buck Semen
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Publisher: 2015 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
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
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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Effect Of Alpha Lipoic Acid On Post Thaw Quality And In Vitro Incubation Of Nili Ravi Buffalo Bull Semen
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Publisher: 2015 Dissertation note: Cryopreservation is the freezing of cells or tissues to subzero temperatures, typically -196 º C. Many benefits have resulted from the process of cryopreservation. Damage induced by cryopreservation has been results cold shock, oxidative stress, osmotic changes, and formation of ice crystal and lipid–protein reorganizations within the cell membrane. Oxidative damage caused by reactive oxygen species (ROS) leads to impaired cell functions. Free radicals, includes ROS and RNS, are normal pro - oxidant molecules in aerobic metabolism. Alpha lipoic acid is a non-vitamin coenzyme that helps in significant metabolic and antioxidant functions in the body. Alpha lipoic acid has been reported to have extra functions by which they are able to synthesize vitamin C from its reduced form in the presence of glutathione. It is matchless among biological antioxidants, because it is equally lipid and water soluble. This allows it to nullify free radicals almost everywhere in the body, inside as well as outside the cells. Therefore, the objective of present study is to determine the effect alpha lipoic acid on post thaw quality and in vitro incubation of buffalo bull semen. Alpha lipoic acid scavenge on reactive oxygen species formed in semen during the process of cryopreservation, so it maintained good semen quality during post thaw and in vitro incubation. Three mature Nili-Ravi buffalo (Bubalis bubalis) bulls (4-8 year age) kept at SPU, Qadirabad Sahiwal Pakistan were used in the study. These bulls are being used as regular donors at SPU. There semen was collected with artificial vagina of temperature 42c; three ejaculates (one from each) was pooled and diluted (30 million sperms/ml) with extender of different inclusion levels (0.0, 0.5, 1, 1.5, 2, 2.5 mmol/ml) of alpha lipoic acid. Straws were filled and extended then semen was cooled for 2 hours and equilibrated for two hours. Semen was placed in Liquid nitrogen vapors for 10 minutes. Finally semen straws was put in liquid nitrogen, Total five replicates were performed. Now post thaw quality was checked in
which various tests were performed, like %age motility, Acridine orange assay for DNA integrity, HOST for plasma membrane integrity, Fitc-PNA/PI for viability and acrosomal integrity. Longevity test was performed by in vitro incubation of frozen thawed semen sample in SOF and evaluating it at 1.5, 3 and 4.5 hour interval in Carbon dioxide incubator. It was expected that Alpha lipoic acid shown positive effect on post thaw quality and in vitro incubation of buffalo bull semen, in the meaning of increased percentage motility, Less DNA damage during cryopreservation and incubation, Increased acrosomal and plasma membrane integrity. So alpha lipoic acid shown positive effect by counter acting on ROS during cryopreservation and in vitro incubation. Results acquired from this study shown that an increase in sperm motility, plasma membrane integrity, DNA integrity, Acrosomal integrity, viability and survival was caused by ALA competences in energy production and anti-oxidant properties, when used at the concentration of 0.5mM and 1mM. In summary, based on the results of our study, it can be concluded that an optimal concentration (0.5mM and 1mM) of ALA improved PMI, sperm motility and viability, minimize DNA damage and improved sperm survival.
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Combine Effect Of Ionomycin And Strontium Chloride To Induce The Parthenogenetic Activation Of Mouse Oocytes
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Publisher: 2015 Dissertation note: Parthenogenesis is a phenomenon in which the development of oocyte oocur without fusion of male gamete. During fertilization spermatozoa trigger intracellular Ca+2 oscllation in M-II stage oocyte which initiates the embryonic development. The rises of intracellular calcium (Ca2+) ions is the basic step for the parthenogenesis. During parthenogenetic activation calcium channel open from endoplasmic reticulnum or depletion of calcium store and facilitate the calcium (Ca2+) from extracellular environment. Parthenogenetic technique is applied in cloning and production of embryonic stem cell lines for used to treat different diseases. Many scientists used different chemicals agents for artificial activation such as strontium, Ionomycin and Ethanol. Strontium chloride has been used widely for parthenogenetic activation of mouse oocyte, but its result to blastocyst development is poor. The objective of present study is to improve parthenogenetic activation and embryo development by combination of Ionomycin with strontium. Hypothesis of my study was Addition of Ionomycin in Strontium based activation protocol improves embryonic development.
The present study was conducted in embryology lab of department theriogenology, university of veterinary and animal sciences, Lahore.Six to eigth week old female mice (n=100) were super ovulated with intra-peritoneal injections of eCG (5iu) followed by hCG injection (5iu) at 48 hrs interval. 14 hrs post hCG, the cumulus oocyte complexes were collected from oviduct of the mice. In experiment 1, the oocytes were activated by using Ionomycin with concentration of 5, 10 and 15 µmol/l for 5 and 10 followed by this activation with strontium chloride (10mmol/l). In experiment: 2, The oocytes were activated by activation medium having strontium (10 mM/l) and Ionomycin (5, 10 or 15 µmol/l) in combination. CZB medium were used for oocyte cultured in CO2 incubator of 5% CO2 at 37°C. Number of activated oocytes were analyzed by cleavage rate to blastocyst stage. In-vitro developmental potential of the activated oocytes were assessed by blastocyst. In experiment: 3, Zygotes were collected 18 h post-hCG and treated with the optimum concentration to check the toxicity effects on embryo development.
In experiment 1, There were insignificant results observed on the bases of cleavage rate in each groups and time of activation as compared to control group. The tendency of morula and blastocysts formation rate was higher (p<0.05) in the 15µM for 10 min activation time as compared to other treatment groups and control group. In experiment 2, The tendency of cleavage rate was significantly higher in the 10 µM and 15µM groups as compared to other treatment group. The blastocyst formation rate was no statistically difference in all treatment and control group. While the toxicity experiment, there was no toxic effect of Ionomycin with Strontium Chloride.
In conclusion, there was higher cleavage rate, 4 cells, morula and blastocyst formation rate in 15µM concentration of Ionomycin for 10 min with Strontium Chloride, there was no toxic effect of Ionomycin with Strontium Chloride on embryos and Ionomycin improved the activation rate and embryo development in combination with strontium chloride.
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Effect Of Alpha Lipoic Acid On Post Thaw Quality Of Jack Semen
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Publisher: 2016 Dissertation note: Improvement of post-thaw quality of Donkey (Equus asinus) semen is essential to augment the in-vivo and in-vitro fertilization rate and to be used for mule production. By the help of cryopreservation sperm cells can be stored for the long time but it causes lethal sub-lethal damage to the sperm. In most species including Donkey and horses sperm cryosurvival rates are not optimal because of its plasma membrane composition. One of the major cryopreservation damage is produced by Reactive oxygen species (ROS) generating oxidative stress caused by ROS are important for normal sperm function but in normal concentration. When they are produced in more quantity they cause damage to Acrosome, DNA and plasma membrane. . Alpha lipoic acid is a non-vitamin coenzyme that helps in significant metabolic and antioxidant functions in the body. Alpha lipoic acid has been reported to have extra functions by which they are able to synthesize vitamin C from its reduced form in the presence of glutathione. It is matchless among biological antioxidants, because it is equally lipid and water soluble. This allows it to nullify free radicals almost everywhere in the body, inside as well as outside the cells. Therefore, the objective of present study was to determine the effect alpha lipoic acid on post thaw quality of jack semen. Alpha lipoic acid scavenge on reactive oxygen species formed in semen during the process of cryopreservation, so it maintained good semen quality during post thaw. Two adult donkeys (Equus asinus) (4-6 years old) kept at animal shed Ravi campus pattoki were used in the study. All the animals were managed under optimal condition of feeding and management. Donkeys were offered green fodder with ad libitum supply of water. Semen collection was done twice a week (one ejaculate/collection) using an equine artificial vagina having temperature of 45-50 ºC. Five collection from each donkey were done (n=10). Ejaculates were filtered with muslin cloth to remove gel. Semen volume was measured by collecting semen in a graduated collection tube after
filtration and the sperm concentration was measured by using a phase contrast microscope (40 x, Nikon) and was scored with a coverslip and then immediately was kept in water bath having 37 ºC temperature after collection until evaluation and processing. Semen quality parameters like volume, concentration and motility were recorded. After initial evaluation, semen samples were extended with centrifugation extender in 1:1 and seminal plasma was removed after centrifugation. Supernatant was removed so that seminal plasma up to 20% will remain with sperm pellet and was maintained at 37 ºC temperature in water bath and was extended with extender having different concentrations of Alpha lipoic acid (0mM, 0.5mM, 1mM, 1.5mM, and 2mM) and cooled for 2 hours and then equilibrated for 2 hrs at 4oC. Then, French semen straws of 0.5ml capacity were filled with semen (100x106/straw). All semen straws were arranged on a rack and then placed at 4cm height above liquid nitrogen vapors for 10 minutes. Finally semen straws was put in liquid nitrogen, Total five replicates per donkey were performed. Now post thaw quality was checked in which various tests were performed, like %age motility, Acridine orange assay for DNA integrity, HOST for plasma membrane integrity, FITC-PNA/PI for viability and acrosomal integrity. It was expected that Alpha lipoic acid shown positive effect on post thaw quality of donkey semen, in the meaning of increased percentage motility, Less DNA damage during cryopreservation and incubation, Increased acrosomal and plasma membrane integrity. So alpha lipoic acid shown positive effect by counter acting on ROS during cryopreservation by reducing melondialdehyde production as indicated by MDA test carried out in this study. Results acquired from this study shown that an increase in sperm motility, plasma membrane integrity, DNA integrity, Acrosomal integrity, viability and survival was caused by ALA competences in energy production and anti-oxidant properties, when used at the concentration of 1.5mM. In summary, based on the results
of our study, it can be concluded that an optimal concentration (1.5mM) of ALA improved PMI, sperm motility and viability, minimize DNA damage and improved sperm survival.
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Effect Of Cholesterol Loaded Cyclodextrin (Clc) Addition On Post Thaw Quality Of Jack Semen
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Publisher: 2016 Dissertation note: Improvement of post-thaw quality of Donkey (Equusasinus) semen is essential to augment the in-vivo and in-vitro fertilization rate. Whenever we think about the techniques for long term storage of the germ cells cryopreservation appears most useful but it causes lethal and sub-lethal damage to the sperm. Sperm cryosurvival rates are not optimal for most species including donkey because of its plasma membrane composition. Egg yolk is an important component of most of the equine freezing extenders. But another factor regarding this important component is that its higher concentrations can have some deleterious effects such as oxidase activity of dead spermatozoa, bacterial/ xenobiotic contamination, reduced respiration and motility of the spermatozoa, which ultimately results in lowering fertility rate. That’s why, cold shock resistance is more in those species semen possessing higher membrane cholesterol to phospholipids ratio as compare to those species semen having less cholesterol to phospholipids ratio. Indeed it may be a useful strategy to improve cryopreservation protocols for jack sperm. Addition of CLC may also be a substitute for egg yolk in semen extender always considering that the cryo-protective effect of EY is partially due to its high cholesterol content. Semen was collected in artificial vagina at 42ºC from two mature donkeys (n=2) five times (replicates=10) which were maintained at Ravi campus Pattoki. Semen samples possessing >60% motility were used in this study. Each ejaculate was divided into 6 aliquots and CLC was added into these aliquots according to desired concentrations (0 mg, 1.5 mg and 3 mg/120 million sperms) then kept at 25ºC for 15 minutes. After incubation semen aliquots were diluted in 1:1with centrifugation media and centrifuged at 600g for 15 minutes to separate seminal plasma after bead formation. Beads of the semen were resuspended in the centrifugation media having different concentrations of egg yolk for different CLC concentration groups in such a way that 100 million / ml sperms concentration was achieved. When this final dilution was done semen was shifted at 4 ºC for 2 hour
cooling then further 2 hours for equilibration at 4º C. Then semen was packed in 0.5 ml plastic straws. These straws were suspended on liquid nitrogen vapors upto7 mints then plunging these straws in the liquid nitrogen in freezing box and then shifting these straws to the liquid nitrogen container by placing them in the goblets and stored there until post thawing was done. After post thawing semen was analyzed for motility, Acrosomal and plasma membrane integrity, live ratios DNA integrity and MDA levels. Post-thaw parameters were analyzed by PROC MIXED as factorial ANOVA using SAS enterprise Guide Version 4.2. And it was observed that. And it was observed that both CLC 1.5 and 3 mg/120 million sperms with full egg yolk did not improved (p˃0.05) the post thaw quality except in malondialdehyde levels in which 3 mg dose with full yolk significantly (p˂0.05) decreased the level of malondialdehyde. While CLC with lower levels of egg yolk maintained the quality same as the control or improved significantly in some parameters.CLC1.5 HEY and CLC3 NEY both compete the control in motility, PMI by HOST and total viability parameter..DNA integrity increased with the decrement of egg yolk as CLC1.5 HEY, CLC3 HEY and CLC3 NEY are significantly (p˂0.05) better than the remaining three groups.MDA level is significantly lower in CLC1.5 HEY and CLC3 HEY groups comparable to control means that with partial removal of egg yolk and supplementation of CLC malondialdehyde levels are decreased significantly as compare to control. Thus CLC substitutes the egg yolk completely when 3mg of CLC /120 million sperms is added in kenney’s extender for jack semen cryopreservation.
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