Bacillus Thuringiensis Toxins Biological Control Aginst Aedes Aegypti
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; Literary form:
Publisher: 2014 Dissertation note: Abstract
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Phylogenetic Analysis Of Major Fresh Water Carps Of Pakistan Through DNA Barcoding
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Publisher: 2014 Dissertation note: Pakistan is bestowed with the land of geological and topographic diversity. The ecological variation is uniformly reflected in all water lands of the country. Pakistan has significantly huge natural inland water resources in the form of ocean, rivers, networks of canals and lakes (Mirza and Rafique 1994). The country is blessed with one of the largest freshwater resources in the world correspondingly large number of freshwater living vertebrates is available from which fishes are quite significant considering the ecological balance and its consumption as food. It is one of the food sources which solely provide all the essential nutrients, minerals and high quality protein which is not common from other food items (Muhammad Rafique 2007). Out of 33,100 fish species identified worldwide as per Fish Base organization report published in April 2015 (http://www.fishbase.org). Out of 233 (indigenous and exotic) freshwater fish species, 78 economically important indigenous fish species are available in the water bodies of the Pakistan. According to this report fishes are the largest vertebrate group, constituting about 50% of all vertebrate species. Systematically fishes are widely spread in nature, ranging from prehistoric jawless fishes to cartilaginous fishes and also from old to current day bony fishes. The taxonomic placement of these fishes shows their belonging to class Actinopterygii, sub-class Teleostei, 3 cohorts, 6 superorders, 13 orders, 30 families and 86 genera (Rafique 2007; Rafique and Khan 2012).
Demand of fish is increasing day by day not only being the naturally available source of food rather the health benefits associated with its consumption. This necessitates to develop a more efficient and sustainable system to increase their growth. DNA based technologies are being competently employed in aquaculture production fields for pedigree information.
Moreover, tagging each fish individually is not an easy task so these DNA based methods help in avoiding intrusion of environmental factors which may result from raising fish families in separate reservoirs (Martinez 2007). Fish identification has been traditionally based on phenotypic features. However, due to high multiplicity and morphological similarity, in many cases, fish at its different developmental stages are hard to be identified by relying only on morphological characteristics (Victor et al. 2009).
For phylogenetic studies of the animals the use of mt-DNA is very common and reliable compared to nuclear DNA due to its high evaluation capabilities, which results in gathering of differences even between closely related species (Moore 1995; Mindell et al. 1997).“Bar-coding gap" is the name given to the property that is inter-specific variation in this region is markedly higher than intra-specific variability (Hebert et al. 2003).
Approximately each and every animal contain 13 protein-coding genes (PCGs) as an essential component of their mt-genome (mitochondrial genome), which helps in encoding of several proteins responsible for the oxidative phosphorylation machinery (Richly A et al. 2004, Song H et al. 2008). Being maternally inherited, mt-DNA is better as compared to genomic DNA such as quick evolution, less exposure to recombination, high copy number, high conservation, little duplications and negligible intergenic regions (Waugh J 2007, Xu J 2005). Clonal inheritance is the main property which makes it more worthy and suitable marker in comparison with other available molecular bio-diversity tools (Galtier et al. 2009).
DNA barcoding is one of the taxonomic tools. It is being used to distinguish animal species based on the small segment of their genome such as mitochondrial DNA, designated as an identification tag or barcode of particular species (Herbert et al. 2003). Identification of species using DNA barcoding is quite debatable. Still many researchers consider it as a reliable
basic tool to ascertain the genetic characterization of diverse eukaryotic species, especially after establishment of the Consortium for the Barcode of Life (CBOL) in 2004 [http: //www. barcodeoflife.org/].
Ideally DNA barcoding should provide quick, reliable and cost effective species identification, even to those user who has little or negligible knowledge of taxonomy (Herbert et al. 2003, Hajibabaei M et al. 2005, Herbert et al. 2005). Identification of unknown source is possible by using distance based tree which can be created by comparing unidentified sequences against retrieved known sequences of different species (Hebert et al. 2003, 2004a, 2004b). DNA barcoding identification system has been recognized universally as standardized method to recognize species and unveil their genetic diversity (Herbert et al. 2003; Herbert et al. 2004). The ideal DNA barcoding is robust, with conserved, universal primer binding sites, reliable DNA amplification and sequencing.
From whole mitochondrial genome, Cytb (Cytochrome b) is considered as one of the most promising gene due to its function and structure, even it is composed of both conserved and rapidly evolving regions which are more related to evolutionary studies (Farias et al. 2001). To identify unknown or ambiguous species it is considered more reliable as it contain sequences which provide the specific information about particular species (Parson W et al. 2000a, 2000b). It is also one of the most useful genetic marker to identify the linkage within families and genera (Meyer 1994; Teletchea 2009). Cytb gene is involved in comparative studies which results in development of new classification schemes and been used to assign a genus to a newly described species as well as improve the understanding of evolutionary relationships of genra (Castresana 2001).
One of the core objectives of this study is to identify and classify four freshwater indigenous fish species of Pakistan, which includes Labeo rohita (Rohu), Labeo calbasu (Calbans), Catla catla (Thalla) and Cirrhinus mrigala (Mori) using Cytb gene. Morphologically, Labeo rohita shows compressed body with convex dorsal profile while mouth bears a pair of barbells and fins are gray and orange in color. Catla catla shows compressed body with broad head. Mouth is wide with thick lower lip. Labeo Calbasu`s dorsal profile is more convex than that of abdomen and two pairs of barbells are present on fringed upper lip. Cirrhinus mrigala has elongated and streamlined body shape which is grayish and silver in color (Bhuiyan AL 1964; Rahman AK 1989). All of these species are found in freshwater bodies mostly lakes, rivers and ponds except Labeo calbasu which is a bottom dweller. These fishes are harvested by using rod and line or by using nets (Talwar PK and Jhingran AG 1991). These fishes are known as major carps and economically very important for the country due to their high consumption as food. These fishes are also used for fish farming due to their greater muscle mass thus also possess very high commercial value for fish farming business.
Another objective of this study is to resolve the taxonomic anomalies related to above mentioned species. Selling of fish meat in mislabeled packaging is a serious issue now days. Most commonly Hypophthalmichthys molitrix (silver carp) and Ctenopharyngodon idella (grass carp) are sold under the label of Labeo rohita. DNA barcoding is also helpful in detecting such fraudulent mislabeling.
It would be the first study in Pakistan to genetically characterize commercially important fish species. It would help scientists to know about their phylogenetic and taxonomic status and also assist fish fanciers to genuinely identify their species of interest. Identification of fish species is also important for conservation of biodiversity as it helps in preservation and
identification of endangered species by generating their barcodes from even minimal evidence available. This study has paved the way for molecular biologists to study taxonomic ambiguities at sub species level using SNP (Single nucleotide polymorphism) based identifying marker.
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DNA Based Characterization of Xylanase Gene From Hyperthermophilic Archeon
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Publisher: 2014 Dissertation note: Blank CD
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Effect Of Adenosine 5’-Triphospate On Post Thaw Quality Of Buck Semen
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Publisher: 2017 Dissertation note: Livestock is the major source of income in agricultural countries. It boosts economic status by producing valuable food products and earning huge amount of Foreign exchange. Pakistan is rich in livestock population having more than 37 goat breeds. Artificial insemination is the most widely used ART and has made the most significant contribution to genetic improvement worldwide. It provides accurate means of progeny testing and spread of genetics to wide area with minimum cost. In contrast to its large scale use in bovine breeding, it is not routinely practiced in small ruminants. It may be due to effects of cryopreservation procedures on sperm plasma membrane, cytoskeleton, motility, mitochondrial membrane, acrosome and the nucleus of spermatozoa, which results in lower fertility rates and discourages the goat breeders to get benefit from this biotechnology. As mitochondria are power house of sperm cell, it provides energy to sperm cell and maintains motility. Therefore, the intactness of mitochondrial membrane must be maintained to keep the sperm cell motile after freezing and thawing procedure. In recent years, a pharmacologic agent, Adenosine 5’-triphosphate (ATP) has been used to enhance the quality and fertility of cryopreserved sperm. Previous reports have shown the freezing of mammalian sperm in hypertonic extenders to improve the rate of sperm viability and acrosome integrity. However, the impact of extracellular ATP supplementation on the quality of goat sperm after freezing and thawing still unknown. Thus the present study was conducted to evaluate the effects of Adenosine 5’ triphosphate (ATP) supplementation in pre-freeze and post thawed buck semen. The semen of six adult Beetal bucks was collected by artificial vagina maintained at 42ºC. After initial evaluation of percentage motility and concentration the semen was pooled and diluted with TCEY extender to the final concentration
of 200 × 106 sperms/mL. The study was conducted in two experiments, in first experiment the extended samples were divided in four equal aliquots and treated with one of the ATP concentrations (0 mM, 0.5 mM, 1 mM, 2 mM). All the samples were incubated at 35 ºC for 15 min. The samples were filled in 0.5 mL French straws cooled to 4ºC within 1 hr, equilibrated at 4oC for a period of 2hr and cryopreserved using standard freezing protocol. After freezing of minimum one week two straws from each aliquot were evaluated for percentages of post thaw motility, sperm viability, acrosome, plasma membrane, mitochondrial and DNA integrities. In second experiment the extended semen samples were cryopreserved with standard freezing protocols. Ten doses were thawed, evaluated individually for post thaw motility and pooled. Now the pooled samples was divided in four equal aliquots and treated with one of the ATP concentrations (0 mM, 0.5 mM, 1 mM, 2 mM). All the samples were incubated at 37 ºC and evaluated at 10 min, 1 hr, 2 hr, 3 hr and 6 hr for percentages of post thaw motility, sperm viability, acrosome, plasma membrane, mitochondrial and DNA integrities. The results of first experiment were analyzed by using one way ANOVA with SPSS. The results indicated that mitochondrial integrity % and DNA integrity % were higher (p < 0.05) in ATP treated groups. Sperm viability % was higher in 0.5 mM group as compared to all other groups. However, post thaw motility %, acrosome integrity % and plasma membrane integrity % remained similar in all groups. The results of second experiment were analyzed using GLM univariate procedure of SPSS. The results revealed that mitochondrial integrity % and DNA integrity % remained higher (p < 0.05) in ATP treated samples throughout the incubation time. Sperm viability % was higher (p < 0.05) in 2 mM ATP group while acrosome integrity % was higher (p < 0.05) in 1 mM ATP group throughout the incubation time. Post thaw motility % and plasma membrane integrity % remained similar (p > 0.05) throughout the incubation time. The interaction between time and
treatment was found non-significant (p>0.05). However, significant (p<0.05) decline in percentages of all parameters was observed with the increasing incubation time. In conclusion, addition of ATP enhances the post thaw quality of buck sperm by maintaining its motility, enhancing sperm viability, mitochondrial and DNA integrities. It also effects positively on post thaw quality of buck sperm. Moreover, is recommended in future to conduct research to elaborate the effects of ATP supplementation in semen on fertility of goats which is also primary objective of cryopreservation of semen.
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Evaluation Of Antimicrobial Activity Of Essential Oil And Extracts Of Nigella Sativa Against Antibiotic Resistant Salmonella Enterica Isolates Of Human And Poultry Origin
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Publisher: 2017 Dissertation note: The research was designed to evaluate the efficacy of methanolic and aqueous extracts of Nigella sativa, Black seed oil and thymoquinone against antibiotic resistant molecular characterized Salmonella enterica isolates of human and poultry origin (n=5 each). The compounds that have shown the antibacterial activity was also checked for their cytotoxicity by MTT assay.
Salmonella is causative agent of invasive diseases in poultry and humans, results in high mortality. Salmonellosis is a disease caused by Salmonella enterica with serious health issues related to food borne illness and most of world’s population is suffering from it. Mostly infections are treated by antibiotics but now a day’s resistance developed by Salmonella enterica. So it is need of time to develop some alternate ways to combat the problem caused by resistant bacterial pathogens. Use of essential oils and extracts of seeds are good weapons against resistant bacteria.
Salmonella enterica isolates of human and poultry origin (n=5 each) were taken from Department of microbiology UVAS Lahore and identified by colony morphology, microscopic characters, biochemical testing (Indole production test, Methyl red test, Voges Proskaeur test, Citrate utilization test and Urea utilization test) and polymerase chain reaction (PCR). For PCR product 1.5% agarose gel was run by gel electrophoresis. The biochemically identified and molecular characterized S. enterica isolates were screened for antibiotic susceptibility by Kirby Bauer disc diffusion method against amoxicillin, ampicillin, cefixime, ceftriaxone, ciprofloxacin, gentamicin, nalidixic acid, co-trimoxazole, ofloxacin and tetracycline and resistant pattern was 100% against ampicillin and Nalidixic acid and isolates shown 60% resistant against co-trimoxazole, amoxicillin and tetracycline, 80% and 40% resistant found against ofloxacin and ciprofloxacin while all isolates sensitive to cefixime and ceftriaxone. Aqueous and methanol were
used as solvents for extraction from Nigella Sativa. Seeds were dried, mixed, centrifuged, filtered and filtrate evaporated to obtained extracts. Percentage yield of methanolic extract was more than aqueous extract. Commercially available black seed oil, thymoquinone, water and methanol extracts of black seed would be evaluated for antibacterial activity by well diffusion method. Zones were measured in millimeters. All compounds gave the zones of inhibition except aqueous extract against Salmonella enterica isolates. The minimum inhibitory concentration (MIC) was determined by micro broth dilution method and then methanolic extract, black seed oil and thymoquinone used in MTT assay to evaluate their cytotoxicity. Cell survival percentage was calculated by a formula.
Data were analyzed by one way analysis of variance (ANOVA) followed by Duncan’s multiple range tests (DMRT) using statistical package for social sciences (SPSS version 20.0). Antimicrobial activity of essential oils, thymoquinone, water and methanol extract would be compared by graph pad prism 5.0 statistical software.
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