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A Study On Superovuation Protocol For The Development Of Embryo Transfer Technique In Mice

by Muhammad Ameen jamal | Dr. Amjad riaz | Dr. Aamir | Prof. Dr Mian abdul sattar.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1900,T] (1).

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Optimization Of Strontium Chloride For Parthenogenetic Activation Of Mouse Oocytes

by Arslan Mahmood Ahmad (2007-VA-67) | Dr. Amjad Riaz | Dr. Aqeel Javeed | Prof. Dr. Mian Abdul Sattar.

Material type: book Book; Format: print Publisher: 2014Dissertation note: There are two main methods by which activation can be performed: (i) physical methods and (ii) chemical methods. Physical methods include electrical stimulation, temperate and mechanical ways, whereas the chemical methods comprise of different artificial chemical agents, including strontium chloride, calcium ionophores, ethanol that promote to rise in intracellular Ca2+ oscillations, cycloheximide, that inhibit protein synthesis and 6-DMAP (6-dimethyl amino purine) which inhibit protein phosphorylation. The contribution of both maternal and paternal genomes is required for thedevelopment to full term of mammalian embryos. However, the percentage of parthenogeneticallyactivated embryos developing to blastocyst stage is lower as compared to normal fertilized embryos. (Renard et al. 1991).In mouse, strontium chloride has been successfully employed in manydifferent studies to induce artificial oocyte activation. The role of strontium to induce calcium oscillations appears to be more physiologically sound than alternativemethods of oocyte activation that produce a monotonic rise in calcium.Strontium chloride (SrCl2) is recognized as one of the most popular parthenogenetic agents for mouse oocytes activation and induces calcium oscillations leads to improved activation rate and blastocyst formation. (Locham-kaplan et al. 2003) (Satoshi et al. 2006). The diploid parthenogenetic oocytes have more developmental competence as compared to haploid form(Liu et al. 2002). A substancecytochalasin B (CB) prevents the release of the second polar body after activation of mammalian oocyte which results in diploid form of embryo (Fukui et al. 1992) and it may also contribute to prevent fragmentation and degradation of embryos ( Yi and Park 2005). Parthenogenetic oocyte activation technique is mainly used in cloning and is a key step for nuclear transfer for cloning. The technique is also useful for understanding of physiological mechanisms of fertilization and early embryonic development. Embryonic stem cells can be derived from fertilized embryos. The stem cells which are produced by parthenogenetic activation have the same totipotency and proliferation as formed by normal sperm-egg fertilization..( Ju et.al 2008). Resultantly, parthenogenetic activation technology has become a target of reproductive biology. This technology can also be used to establish embryonic stem cell lines (Mizutani et al. 2004) and embryonic stems cells are the fundamental source in field of regenerative medicine; used to treat many diseases such as diabetes, beta thalassemia, heart infarction etc by providing patient specific replacement cells. Mouse is one of the most commonly animal models used for parthenogenetic activation. The other animals which have been used for parthenogenetic activation include rabbits, cattle, sheep, horses, monkeys and pigs. Parthenogenetic embryos are failed to develop to term, due to genomic imprinting, an epigenetic change of certain genes, depending on the parent of origin.(Uranga and Arechaga 1997). The studies pertaining to parthenogenetic activation technology for mouse oocytes is extremely limited at present (Mizutani et al. 2004). Availability: Items available for loan: UVAS Library [Call number: 2188,T] (1).

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Comparison Between Aspiration And Slicing Methods For Retrieval Of Oocytes In Bovine

by Muhammad Husnain (2008-VA-281) | Prof. Dr. Mian Abdul Sattar | Dr. Qaiser Shahzad | Dr. Amjad Riaz | Dr. Muhammad Avais.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation 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. Summary 26 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. Availability: Items available for loan: UVAS Library [Call number: 2279-T] (1).
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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

by Khalid Mahmood (2005-VA-114) | Dr. Amjad Riaz | Prof. Dr. Mian Abdul Sattar | Dr. M. Hassan Saleem.

Material type: book Book; Literary form: not fiction Publisher: 2016Dissertation 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 embryo production. 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 CHAPTER 6 SUMMARY 38 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 cattle. Summary 39 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 Pakistan. Availability: Items available for loan: UVAS Library [Call number: 2636-T] (1).

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