Comparative Effect Of Alpha Lipoic Acid And Butylated Hydroxytoulene On Post Thaw Quality Of Buck Semen
Material type: Book ; Literary form:
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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Comparison Of Commercial Triladyl Extender With A Tris-Citric-Egg-Yolk (TCEY) Extender On Post-Thaw Semen Quality Of Nili Ravi Buffalo
Material type: Book ; Literary form:
Publisher: 2016 Dissertation note: Cryopreservation of semen is the most important step for its usage in artificial insemination. Freezing of semen leads to a remarkable reduction in post-thaw semen quality. Therefore, selection of a better semen extender has always been considered priority that could serve as a good cryoprotectant.. Our semen production units (SPUs) have been using Tris based egg yolk semen extender since long time. Some modern SPUs like CEBG are using commercially available semen extenders for better post-thaw semen quality.
After collection pooled semen divided into two equal aliquots in separate sterilized test tubes and kept in water bath at 37 ºC. Semen was diluted with each of extender (TCEY and Triladyl) on the basis of sperm concentration (40x106sperm/ ml). Diluted semen was placed bottles and placed in safety cabinet cooled to 4 ºC over and equilibrated for 4 hrs. After equilibration semen was filled in 0.5 ml French straws (20x106sperm/ 0.5 ml). All semen straws placed in automatic freezer 4cm above liquid nitrogen surface in vapors for 10 minutes. Liquid Nitrogen vapors used in automatic programmable freezer to reduce temperature from 4 ºC to -180 ºC and then plunged into liquid nitrogen -196 ºC for freezing and was stored until analyzed. The experiment was repeated for seven times (replicates = 07)
CASA sperm motility parameter and kinematics were analyzed at Center of Excellence for Bovine Genetics (CEBG) Renala khurd District Okara. For further analysis frozen semen straws were brought to the Department of Theriogenology UVAS, Lahore. Effects of Triladyl and TCEY on post-thaw semen quality of the Nili Ravi buffalo semen were compared.
In Triladyl group, significantly (P<0.05) higher post-thaw motility (PTM %), Plasma membrane integrity (PMI, %),) DNA integrity (%), Live percentage was found. However, no significant (P<0.05) difference was found regarding NAR results between both groups. Sperm abnormalities were found significantly lower in Triladyl group as compared to TCEY group.
In overall assessment regarding and post-thaw CASA motility parameters, CASA motility, (PROG %), rapid (RAP %), medium (MED%), and slow (Slow, %) and sperm motility kinematics (VAP μm/sec), (VSL μm/sec), (VCL μm/sec), (ALH μm), (BCF HZ), (STR%) and (LIN%) Triladyl was found better than TCEY.
This was concluded that use of commercial semen extender Triladyl resulted in significantly better post-thaw semen quality as compared to Tris citric egg yolk (TCEY) extender.
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Comparison Of Estradiol Benzoate And Gnrh In Cidr Based Superovulation Protocols For The Initiation Of Follicular Wave Emergence In Exotic And Crossbred Cattle
Material type: Book ; Literary form:
Publisher: 2016 Dissertation note: Background: Livestock is a major contributor to the national (11.9%) and agriculture (55.4%)
economy in Pakistan. Milk and meat are major livestock products of Pakistan, which is ranked
fourth largest milk-producing country in the world. The growth rate of dairy sector is growing
very fast for last several years, however, the genetic potential of elite cows is continuously
deteriorating due to unavailability of reproductive biotechnologies such as embryo transfer.
Normally one calf per year can be obtained from elite mothers. Maximum number of offspring
can be obtained by superovulation. This will helpful in production of genetically superior
offspring in limited time thus resulting in maximum exploitation of genetic potential of elite cows.
In Pakistan more than 80% farmers are small holder having 2-3 animals with low genetic
potential. The use of elite mothers by these farmers is limiting due to high cost. Superovulation
is a strategy that can be used to make low cost embryos available for small holders. This will
result in maximum spread of genetic potential of superior females. Use of follicular wave
emergence based super stimulation and timed ovulation with help of CIDR can improve the
results of super ovulation protocols and may be an effective tool to improve the per unit time
Hypothesis: Use of EB or GnRH in CIDR based superovulation protocols may result in
improved super ovulatory response in cattle.
Methodology: This study was conducted at Centre of Excellence for Bovine Genetics Embryo
Transfer Wing Okara. Seventy Donor cows (mix of crossbred and Holstein Frisian) were selected.
Animals coming into natural heat, were randomly assigned into one of the following
superovulation protocols; (A) In first group (n=37), which was considered as control, on 8th day
after heat animals were palpated for presence of a good quality CL and super ovulatory treatment
i.e. twice daily FSH injection were started on “Day 11” of its cycle for four consecutive days. On
day 3rd of FSH treatment PGF2α was injected both in the morning and evening. Animals were
inseminated in the morning and evening on the 5th day of superovulation treatment and next day
morning based on detected heat. Embryos were collected from the animal on 7th day after first
insemination. A PG injection was administered to the animal three days after embryo collection.
(B) In second superovulation protocol (n= 15), the animals were palpated for the presence of a
good quality CL on “Day 8” and a CIDR was placed after the confirmation of CL. An injection
of 2mg EB (Estradiol Benzoate) was also administered on the same day. Super ovulatory
treatment was initiated by “Day 11” of its heat cycle as narrated in first superovulation protocol
with only difference of CIDR removal along with 7th dose of FSH. Animals were inseminated
with a single straw of semen if on heat in the next day morning (Day 16 after natural heat) or with
double straw at “3 pm” if not on heat in the morning. Animals were also inseminated on next day
morning if heat sustained till next day morning. Animal were collected by non-surgical flushing
seven days after first insemination at super estrus. A PG injection was also given to the animals
three days after embryo collection. (C) In third superovulation protocol (n=18), the animal were
palpated for the presence of a good quality CL on “Day 8” and a CIDR was placed after the
confirmation of CL. An injection of “2ml Dalmaralin” was administered on the same day. Super
ovulatory treatment was initiated by “Day 11” of its heat cycle in similar sequence as narrated in
second superovulation protocol. At least 5 animals of each category (i.e. Crossbred and Holstein
Frisian) were treated with each super stimulatory protocol. In conclusion, CIDR plus GnRH or
CIDR plus EB protocols are better than normal superovulation protocol for embryo production in
Outcomes: This study remained helpful to improve the existing superovulation protocols for
bovines with promising results which will help the genetic improvement programs of bovine in
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