Standardization Of Bovine Serum Albumin (Bsa) For Cryopreservation Of Beetal Buck Semen
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Publisher: 2014 Dissertation note: Abstract
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Comparative Effect Of Cidr Based Estrus Synchronization Protocol With Or Without Gnrh In Non-Descript Cows During Low Breeding Season
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Publisher: 2015 Dissertation note: The livestock sector occupies a peculiar position in the national plan of economic development in Pakistan. It subscribed approximately 55.9 percent to the agricultural value added and 11.8 percent to national GDP with a growth rate of 2.7 % in 2013-14 (Anonymous 2014). Dairying has become an important subsidiary source of income for thousands of rural families with an important role in generating earning opportunity. In Pakistan dairy sector is developing and commercializing at a rapid pace to meet increasing requirement of milk and other dairy products (Dongre et al. 2011).
Pakistan owns renowned breeds of dairy buffaloes (Nili-Ravi and Kundi) and cattle (Sahiwal, Red Sindhi, Thari and many others). Cattle in Pakistan belong to genus Bos indicus. According to the latest livestock census (Anonymous 2006), out of 29.6 million cattle, 46% (13.6 million) have been described as non-descript. Non-descript cattle do not fall in any defined breeds of cattle. Milk production of non-descript cows is < 1000 lit per lactation in mountains area of NWEP pakistan (Khan and Usmani 2005). As non-descript breeds make up the largest group of cattle in Pakistan, there is a dire need to work on genetic improvement of these animals. Artificial insemination, the best tool for genetic up-gradation in dairy cattle is applied only in 11.1% cows in Pakistan (Anonymous 2006). The main hindrances are small sized scattered herds and lack of experienced technical manpower in the field. Estrus synchronization of a large number of animals and timed insemination can be used to overcome these hindrances .The technique may also help in
reducing a prolonged calving interval and postpartum anestrus and seasonality of breeding in these animals (Zafar et al. 2008).
Estrus synchronization widely practiced in temperate dairy cattle in developed countries (Hansen and Arechiga 1999). Before launching a large scale estrus synchronization program in non-descript cattle, there is a need to assess the efficacy of various synchronization protocols in terms of estrus incidence, intensity and conception rate. Additionaly, distinct differences have also been reported between Bos taurus and Bos indicus in terms of estrus duration and intensity of expression of estrus sign (Mattoni and Ouedraogo 2000). The low estrus intensity and less duration of estrus signs of Bos Indicus are due to smaller diameter of follicle as compared to that of Bos Taurus (Bo et al. 2003).
Developing successful methods for synchronizing estrus and ovulation in cattle has been a major research interest. Ultimately, the goal has been to achieve precise synchronization of ovulation so that cattle can be inseminated without regard to estrus detection. One method to increase conception rates is to use hormonal treatments in zebu breeds for synchronizing ovulation and for timed artificial insemination (TAI). Hormonal programs for synchronizing ovulation to control the follicular and luteal phases and estrus behavior have been used in Bos taurus cows and heifers(Castellanos et al. 1997), and Bos Indicus cows (Pinheiro et al. 1998).
The intensity and duration of estrus behaviors during the estrous cycle is highly variable among individuals. More commonly animals are diagnosed to be in estrus based on the mounting or standing to be mounted, appearance of mucus discharge, and other physical activities (Van Eerdenburg et al. 2002). Scoring system were established on the basis of observed estrus signs and most of them have taken mounting and standing to be
mounted behavior as the most reliable signs to predict ovulation time in Bos taurus cows (Roelofs et al. 2005). However, it is now well acknowledged that the expression of estrus behavior change with breed of cows (Naidu and Babu Rao 2006).
Fertility is an important parameter to assess the efficacy of estrus synchronization. Bos indicus cows after treatment with CIDR conception rate in adult cows 40% and in heifers 20% (Singh et al. 2006). CIDR may have ability to overcome the problems in field condition and increasing the reproductive efficiency by minimize the hindrance anestrus postpartum cows. In Pakistan research work have been done to evaluate the efficacy of CIDR for conception rate in indigenous cattle. Until now no study conducted on non- descript cattle. Therefore, it is hypothesized that CIDR+GnRH can provide better in vivo fertility compared to CIDR.
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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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Comparison Between Aspiration And Slicing Methods For Retrieval Of Oocytes In Bovine
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Publisher: 2015 Dissertation note: Livestock contribution to agriculture stood at 55.9 percent while it contributes 11.8 percent to the national GDP during 2013-14. Buffalo, cattle, sheep and goat population in Pakistan is 34.6, 39.7, 29.1 and 66.6 million numbers during 2013-14. Total milk production from buffalo and cattle as major milk producing animals is 31,252 and 18,027 (000 tons) (Economic Survey of Pakistan 2013-14).
Advanced biotechnologies coming from different areas of biological sciences exhibit great promise to enhance the efficiency of livestock production. From these technologies one such biotechnology is the use of in vitro maturation of follicular oocytes and in vitro fertilization for production of livestock embryos in laboratory. Proper oocytes recovery and their selection in the laboratory are of great importance for successful in vitro embryo production. Total one hundred and forty four ovaries (n=144) from cattle (72 ovaries) and buffalo (72 ovaries) were collected and 223 oocytes were retrieved from these ovaries. Average oocytes per ovary were 1.66 + 0.43 oocytes per ovary were obtained via aspiration and 1.89 + 0.00 average oocytes per ovary through slicing method from cattle ovaries. Average 1.55 ± 0.55 oocytes per ovary via aspiration and 1.53 ± 0.20 oocytes per ovary through slicing from buffalo ovaries.
Overall grade-A oocytes were 28 (40) percent with aspiration in cattle and 25(36.76) through slicing method. In buffalo overall grade-A oocytes retrieval was obtained in percentage as 20 (44.44) and 26 (52) through aspiration and slicing methods respectively. Grade-B oocytes recovery obtained was in percentage as 23 (33.82) with slicing and 19 (31.67) through aspiration technique from cattle ovaries.
Commonly used methods of recovery of oocytes from slaughterhouse animals are aspiration and slicing. Recovery rate of oocytes is different from slaughterhouse ovaries. Aspiration is the best method for retrieval of good quality oocytes from slaughterhouse bovine ovaries because it gave more good quality oocytes in less time than slicing method. In this study, it is found that weight of ovary and no. of follicles/ovary in cattle have strong correlation of 71% existed between weight of ovary and no. of follicles /ovary in buffalo was observed. Correlation between average number of follicles on ovary and weight /ovary was stronger in cattle. The more the number of follicles present on the ovaries and more weight of the ovary, the more will be the recovery of oocytes.
In cattle average number of follicles was 10.09 ± 0.30 and when it was checked in buffalo, differed significantly and it was found as 7.16 ± 0.19 on an average per ovary. Likewise weight of buffalo in this study was differed significantly from cattle 4.04 ± 0.10 and 7.62 ± 0.15 respectively. It is suggested that oocytes retrieval should be done in buffalo using aspiration method to retrieve better quality oocytes.
It is concluded that aspiration is the suitable method for retrieval of good quality oocytes from slaughterhouse buffalo ovaries because it gave more good quality oocytes in less time than slicing method. But both methods have minor difference between recovery rates but aspiration is more convenient than slicing and it yields more quality oocytes. It is also found that there is very strong correlation existed between average weight of ovary and number of follicles per ovary and the both parameters play a great help for more quality and quantity oocytes.
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Effect Of L-Cysteine And Glutathione On Post Thaw Quality Of Sahiwal Bull Spermatozoa
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; Literary form:
Publisher: 2015 Dissertation note: Freezing and thawing of semen leads to production of reactive oxygen species (ROS)
due to plasma membrane lipid peroxidation. Because of this semen quality can be
compromised. To overcome this problem, antioxidants have been used in cryopreservation
medium. Glutathione and cysteine have thiol groups which penetrate into the cell and protect
it from oxidative stress. In this study, effect of different concentrations of cysteine and
glutathione on post thaw quality of Sahiwal bull spermatozoa was determined.
Semen was collected with artificial vagina from five mature regular donor Sahiwal
bulls kept at the Semen Production Unit Qadirabad, Sahiwal. Semen samples possessing
>60% motility and >500x10
sperm/ml were included in study. After collection, semen
samples from five bulls were pooled, divided into seven equal aliquots and kept at 37 ºC in
water bath. After that dilution was done with Tris citric egg yolk extender having different
concentrations of cysteine and glutathione as Con (0.0 mM), C1 (1.0 mM cystein), G1 (1.0
mM glutathione), CG0.5/1(0.5 mM Cysteine+1.0 mM glutathione), CG1/0.5 (1.0 mM
cysteine+0.5 mM Glutathione), CG0.5/0.5 (0.5 mM cysteine+0.5 mM glutathione) and
CG1/1 (1.0 mM cysteine+1.0 mM glutathione). Diluted samples were cooled to 4ºC in two
hours and equilibrated for 4 hours at 4
C. After that they were packaged into 0.5 ml French
semen straws (20x10
sperm/straw). All semen straws were placed 4cm above liquid nitrogen
surface in vapors for 10 minutes. Then, semen straws were plunged into liquid nitrogen for
freezing and stored until post thaw analysis. The experiment was repeated for five times
(replicates = 5). Four semen straws/treatment were thawed for 30 seconds in water bath at
37ºC and evaluated for visual motility, plasma membrane integrity (PMI), acrosome integrity,
mitochondrial trans membrane potential and CASA motility parameters and kinematics.
PMI in group CG0.5/0.5 was significantly higher (40.00±1.42 %) as compared to Con
26.67±0.80 (P<0.5). Plasma membrane integrity in groups CG1/1, CG0.5/1, G1 and C1 was
significantly higher (36.00±1.88 %, 36.20±1.07 %, 33.60±1.21 % and 32.80±0.80 %
respectively) as compared to Con (26.67±0.80 %) (P<0.05). There was no significant
difference in C1 (32.80±0.80 %) and G1 (33.60±1.21 %) (P>0.05). In case of acrosome
integrity, NAR value of group CG0.5/0.5 was significantly higher (71.40±1.08 %) as
compared to Con (59.67±0.37 %) (P<0.05). All other groups also showed significant
differences as compared to Con (P<0.05). CG0.5/0.5 also showed significantly higher NAR
value (71.40±1.08 %) as compared to C1 (64.40±1.40 %) and G1 (67.60±2.07 %) (P<0.05).
CG0.5/0.5 had significantly higher value (71.40±1.08 %) as compared to CG1/0.5 and CG1/1
(65.60±0.81 % and 68.80±0.97 % respectively) (P<0.05). CG0.5/0.5 had significantly higher
subjective motility (54.00±1.88) as compared to Con (36.66±0.92)
Mitochondrial transmembrane potential of CG0.5/0.5 was significantly higher
(37.00±0.71 %) as compared to Con (25.33±1.28 %) (P<0.05). All the other treatment groups
also had higher mitochondrial transmembrane potential as compared to Con (P<0.05). In
groups of combination of cysteine and glutathione, CG0.5/0.5 showed significant difference
(37.00±0.71 %) as compared to CG1/1 and CG1/0.5 (29.00±1.00 % and 33.80±0.86 %)
CASA results showed that CG1/1 had significantly higher motility as compared to the
control. But the percentage of progressive spermatozoa was significantly higher in
CG0.5/0.5. VSL of group CG0.5/0.5 was significantly higher (53.33±2.90 %) as compared to
Con (45.10±0.50 %). However, VSL, VCL, ALH and BCF did not vary significantly among
groups. STR and LIN of group CG0.5/0.5 were significantly higher as compared to the
In conclusion, addition of cysteine and glutathione in tris citric egg yolk extender
improved the post thaw quality of Sahiwal bull spermatozoa. In case of additive effect of
cysteine and glutathione, CG0.5/0.5 showed higher plasma membrane integrity, acrosome
integrity, mitochondrial transmembrane potential, progressive and rapid spermatozoa as
compared to CG0.5/1, CG1/0.5 and CG1/1.
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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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Effect Of Β-Carotene And Tocopherol On Pregnancy Rate In Cidr Synchronized Nili-Ravi Buffaloes
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Publisher: 2017 Dissertation note: Buffalo is of unique importance in livestock and dairy industry of Pakistan due to its high milk production and shares 65% of total milk production. Reproduction is important to get good production and profit in dairy sector. Reproduction in buffalo is compromised due to its small size ovaries, poor ovarian reserves and less pronounced estrus intensity, resulting as low fertility. Synchronization techniques including CIDR based protocols are well established in cows and getting popularity in buffaloes but with low results, comparatively. Therefore, some modifications are required based on physiology of estrus cycle in buffalo. This was hypothesized that additional injection of Dalmavital in CIDR base protocol will enhance the pregnancy rates and embryonic liability by minimizing the oxidative stress. Therefore, present study is conducted to evaluate the effect Dalmavital on estrus response, Estrus intensity, Pregnancy rates and embryonic losses in CIDR synchronized Nili-Ravi buffalo. For this, buffaloes were scanned ultrasonically for the reproductive tract evaluation. Reproductively sound buffaloes were selected and randomly allocated to one of the two treatment group. 86 buffaloes with normal reproductive tract were assigned in two groups 1; CIDR group (n = 43) and 2; CIDR+D (n = 43). AI was performed twice at 48 and 60 hours after CIDR removal. Estrus response (ER) did not differ significantly (P >0.05) in groups, CIDR and CIDR-D but estrus intensity (EI) was statistically significant (P<0.05) in treatment group. Pregnancy rates were also non-significant (P>0.05) in treatment and control group but improved comparatively in CIDR-D group (63% in CIDR-D group and 56% in CIDR group). Embryonic and fetal losses were also non-significant (P>0.05) between the control and treatment group. Results were also compared in cyclic and non-cyclic, Milking and dry, BCS and parity. Results were non-significant in milking and dry, BCS and Parity. Pregnancy rates were found different significantly (P<0.05) in cyclic and non-cyclic animals, when treatment
is ignored. From the present study it can be concluded that Dalmavital may have good effect on estrus intensity in CIDR synchronized Nili-Ravi buffalo.
Nili-Ravi buffalo is known as black gold of Pakistan. They produce about 2500 liters of milk with 6.5% butter fat. Despite of benefits, this breed is highly influenced with low reproductive activity that include prolonged pubertal period, poor exhibition of estrus, inadequate ovarian activity, long calving interval, high embryonic mortality and low fertility rate with artificial insemination. These factors reduce buffalo’s reproduction which leads to great economic losses. Therefore, there is dire need to address these problems and to orchestrate novel approaches to enhance the reproductive efficiency of buffalo. In cows, during last six to decades, researchers have considerably devised certain strategies to synchronize estrus with the help of prostaglandins, progestagens and estrogens. The advent of these hormones or synchronization protocols helped significantly in improving reproductive management. Moreover, the use of fixed time artificial insemination protocols resulted in acceptable fertility. However, these tools provide inconsistent results to manage reproduction in buffalo. Therefore, unprecedented approaches are required to facilitate and improve the buffalo reproduction.
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