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Anand Agricultural University, Anand
Anand Agricultural University (AAU) was established in 2004 at Anand with the support of the Government of Gujarat, Act No.(Guj 5 of 2004) dated April 29, 2004. Caved out of the erstwhile Gujarat Agricultural University (GAU), the dream institution of Sardar Vallabhbhai Patel and Dr. K. M. Munshi, the AAU was set up to provide support to the farming community in three facets namely education, research and extension activities in Agriculture, Horticulture Engineering, product Processing and Home Science. At present there seven Colleges, seventeen Research Centers and six Extension Education Institute working in nine districts of Gujarat namely Ahmedabad, Anand, Dahod, Kheda, Panchmahal, Vadodara, Mahisagar, Botad and Chhotaudepur
AAU's activities have expanded to span newer commodity sectors such as soil health card, bio-diesel, medicinal plants apart from the mandatory ones like rice, maize, tobacco, vegetable crops, fruit crops, forage crops, animal breeding, nutrition and dairy products etc. the core of AAU's operating philosophy however, continues to create the partnership between the rural people and committed academic as the basic for sustainable rural development. In pursuing its various programmes AAU's overall mission is to promote sustainable growth and economic independence in rural society. AAU aims to do this through education, research and extension education. Thus, AAU works towards the empowerment of the farmers.
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ThesisItem Open Access EXPRESSION PROFILING, SNP DETECTION AND VALIDATION IN SQUAMOUS CELL CARCINOMA OF HORN IN KANKREJ CATTLE (Bos indicus) USING NEXT GENERATION SEQUENCING(AAU, Anand, 2014) KORINGA, PRAKASHKUMAR G.; Joshi, Chaitanya G.Horn cancer is a widely prevalent cancer amongst Kankrej cattle (Bos indicus) seen sporadically, especially in case of working class of castrated male animals i.e. bullocks. A transcriptome envisaged characterization as well as correlation to known genomic changes such as structural and copy number alterations, focused ins/dels and single nucleotide mutations. Here, we employed high throughput RNA-seq using GS-FLX Titanium for characterization and comparison of normal and cancerous horn transcriptome in Bos indicus. A total of 909,362 reads with average read length of 405bp for horn cancer (HC) and 583,491 reads with average read length of 411bp for horn normal (HN) were obtained by sequencing gene transcripts derived from HC and HN tissues. Assembled data were analyzed for identifying novel as well as differentially expressed transcripts using CLC Genome Workbench. RNA-seq analysis using different bioinformatics pipelines and software identified differentially expressed genes i.e. upregulation of KRT6A, KRT6B, KRT6C, KRT14, SFN, KRT84, PI3, CAl, C0L17A1, ANLN, SERPINB5 etc., as well as down-regulation of NR4A1, FOSB, LRIGl, BOLA, SCGBIAI, CXCL17, KRT19, BPIFBl, NR4A1 and TFF3 etc., in HC tissues. The signaling pathway investigation in this study revealed many of the cancer related pathways which mainly include cell cycle regulation pathways, p53 tumor suppressor pathways, NFKB and MAPKs pathways, LPS signaling pathway and PI3K-Akt pathways. The resuh of transcriptome expression profiling was validated using RT-qPCR in nine randomly selected genes. It revealed concordance of gene expression profile with RNA-seq analysis. We also used transcriptome data to elucidate complexity of the alternative splicing in HC transcriptome. We identified potential candidate splice variants that might be helpful in development of relevant biomarkers for early diagnosis of HC. The fiiture studies targeted at in depth characterization of these potential candidate splice variants might change the currently used clinical approaches. Herein we characterized global landscape of alternative splicing events exhibited by pair of HC and HN tissue and confirmed selected alternative splicing events with significant association to HC by RT-qPCR. Ine analysis of the same RNA-seq data using SeqMan Pro Version 10.0.0 resulted in to a 9532 and 7065 SNPs as well as 1171 and 1172 Indels in HC and HN, respectively. Out of total, 7889 SNPs and 1736 Indels uniquely present in HC, 5886 SNPs and 1146 Indels uniquely present in HN are novel and reported first time in Bos indicus, whereas rest are already reported in Bos taurus dbSNP database at NCBI. The gene-associated SNPs and Indels were high in upregulated genes of HC as compared to HN tissues. SNPs identified in RNA-seq analysis were validated in fiirther studies in two groups consisting of 50 animals each of HC and HN bullocks. DNA from HC tissue and blood of HN individual was extracted and 96 pairs of primers were used to generate amplicons of an average 300bp to get sequenced using Ion Torrent PGM. The resulting reads were assembled using SeqMan N Gen of DNASTAR and data were analyzed using Arraystarll. Case control analysis was carried out to find SNP significantly associated with HC. SNP at position 63251805 (dBSNP ID rsl36870681) identified in BPIFAl can serve as a potential candidate genetic marker in HC. The SNPs and Indels identified in this study will be useful resource for future studies to understand genetic basis for phenotypic variation between Bos taurus and Bos indicus as well as cancers in animals. A very large number of SNPs are essential for the designing and construction of arrays. SNPs identified in this study will enrich the dbSNP database of NCBI (http://www.ncbi.nlm. nih.gov/projects/SNP/) and will be useful resource for array designing. This study is the first attempt to reveal novel transcripts, differentially expressed genes as well as identification and validation of SNPs using digital expression analysis in Bos indicus and provides novel insights into bovine transcriptome. Our study will serve as a step further in detailed characterization of HC transcriptome and provide firm base to explore and mitigate HC at finer resolution. The present findings would provide basis for further screening of genes and identification of markers for early diagnosis and therapeutic intervention of HC.ThesisItem Open Access REGULATION OF ACTIVIN TYPE II RECEPTOR B (ACVR2B) EXPRESSION THROUGH RNA INTERFERENCE IN GOAT MYOBLAST CELLS(AAU, Anand, 2014) PATEL, AMRUTLAL KALUBHAI; Joshi, Chaitanya G.Enhancement of skeletal muscle mass through genetic manipulation has drawn attention to increase the meat production in the farm animals. Among the various techniques of regulating gene expression, RNA interference (RNAi) has been proposed as a promising tool to suppress the target gene expression. Attempts have been made to increase the skeletal muscle mass in transgenic animals through knockdown of Myostatin, a gene with potential negative effect on muscle growth. It has been well established that myostatin mediates its action through binding to its cell surface receptor mainly to activin type II receptor B (ACVR2B). Besides regulating myostatin activity, ACVR2B has also been known to regulate the activity of other Transfonning Growth Factor beta (TGF-(3) superfamily ligands which negatively regulates muscle growth. The inhibition of ACVR2B signaling has shown dramatic increase in the muscle mass to a greater extent than myostatin inhibition. Hence, in the present study we aimed to investigate the possibility of ACVR2B knockdown to enhance the myogenesis in goat through various RNAi methods such as expression of short hairpin RNAs (shRNAs) under U6 and CMV promoters and expression of artificial microRNAs (amiRNAs) under CMV and muscle specific muscle creatine kinase (MCK) promoters. Further we studied effect of ACVR2B knockdown on the expression of myogenic regulators and assessed induction of undesired interferon response against RNAi vectors. Among the seven shRNAs tested, the U6 promoter driven shRNAs showed 63 (p= 0.0004), 76 (p= 0.0001), 75 (p=0.0000), 74 (p= 0.0005), 80 (p= 0.0001), 74 (p= 0.0000) and 57% (p= 0.0013) silencing with sh1 to 7, respectively in HEK293T cells whereas 24 (p= 0.1497), 24 (p= 0.2243), 15 (p= 0.3988), 31 (p= 0.1263), 14 (p= 0.4425), 46 (p= 0.0318), and 26 % (p= 0.1288) silencing of endogenous ACVR2B with shl to sh7, respectively and 53 (p- 0.0005), 32 (p= 0.0171), 38 (p= 0.0025), 66 (p= 0.0002) and 51% (p= 0.0008) with sh3, sh4, sh5, sh6 and sh7, respectively against ectopically expressed goat ACVR2B in goat myoblasts ceils. The knockdown of endogenous ACVR2B resulted in the 116, 105, 84, 64, 119, 102, 121. and 157% expression of MyoD; 131, 128, 128, 123, 104, 103, 69, and 157% of MyoG in sh1 to sh7 transfected cells, respectively. The transfection of U6 driven shRNA resulted in the induction of OAS1, a marker for innate interferon response by 3 to 1861 fold in 293T cells and up to 94 fold in the goat myoblasts cells. In an attempt to overcome the undesired cellular toxicity associated with U6 driven shRNAs as reported in the number of studies, we expressed these shRNAs under CMV promoter. The CMV driven shRNAs showed weak silencing of 37 (p= 0.1622) and 18% (p= 0.4877) by sh1 and sh3, respectively in HEK293T cells whereas 7% (p= 0.5749) by shl and 4% (p= 0.7493) by sh5 in goat myoblasts cells. Unlike suggested earlier, we observed significant induction of interferon response to CMV driven shRNAs up to 46 fold in 293T cells and 105.3 fold in goat myoblasts cells. Alternatively, we assessed another RNAi approach using amiRNAs which mimics the endogenous miRNA biogenesis pathway. Among the four amiRNAs tested by placing them in 5'-UTR region of GFP reporter, we observed 64 (p=0.0004), 77 (p=0.0002), 1 (p=0.8712), and 41% (p=0.0115) silencing in 293T cells by ami204, ami318, ami735 and ami878, respectively against exogenously expressed goat ACVR2B; 19 (p=0.3593) and 9% (p=0.4977) by ami204 and ami318, respectively against endogenous ACVR2B and 23% (p=0.0444) by ami318 against exogenously expressed ACVR2B in goat myoblasts cells. Since, amiRNAs placed in 5'-UTR were shown to affect the translation of reporter GFP, we further placed them in 3'-UTR of GFP which resulted in enhanced expression of GFP thereby enabling the monitoring of expression of amiRNAs. The 3'-UTR derived amiRNAs showed 50% (p=0.0002) silencing only by ami3I8 in 293T cells whereas 47 (p=0.0193), 16 (p=0.2959), 19 (p=0.1547), and 28% (p=0.0770) by ami204, ami318, ami735 and ami878, respectively against endogenous and 67%) (p=0.0004) by ami318 against overexpressed ACVR2B in goat myoblasts cells. The expression of myogenic regulators MyoD remained unchanged by amiRNAs cloned in the 5'-UTR whereas expression of MyoG was significantly up regulated by ami878 (p=0.0089). However, amiRNAs cloned in 3'-UTR showed significant down regulation of MyoD by 51 (p=0.0007), 27 (p=0.0232), 29 (p=0.0074), and 31% (p=0.0104) and MyoG by 36 (p=0.0034), 12 (p=0.2532), 22 (p=0.0303), and 37% (p=0.0026) by ami204, ami318, ami735 and ami878, respectively. As observed for U6 and CMV driven shRNAs, CMV driven amiRNAs showed significant induction of interferon response in 293T (up to 121.7 fold) and myoblasts (212.5 fold) cells. As ACVR2B has been shown to be essential for embryonic development, we tested the possibility of its knockdown in skeletal muscle using muscle specific MCK promoter. Among the MCK and MSTN promoters with and without two repeats of MCK enhancer, we observed maximum transcriptional activity by MCK promoter in goat myoblasts cells. We thus tested best amiRNAs (ami204 and ami318) by expressing under MCK promoter which showed 22% silencing efficacy by ami318 in 293T cells and 32% silencing efficacy in goat myoblasts cells by transient transfection assay. Further to test the possibility of ACVR2B knockdown after stable integration of amiRNAs into goat myoblasts genome, we generated lentivirus particles carrying amiRNAs expression cassettes and transduced the goat myoblasts. The myoblasts cells stably integrated with amiRNAs showed ~8% silencing by ami318 which was increased to 34% upon induction of differentiation under muscle specific promoter. Western blot analysis revealed 41% and 57% silencing by 5'-ami204 and 5'-ami318, respectively whereas 14% and 35% silencing by 3'-ami204 and 3'-ami318, respectively in stable myoblasts upon induction of differentiation. Unlike transient transfection assay vv'hich showed positive correlation of expression of ACVR2B with myogenic regulators, its stable knockdown resulted in up regulation of MyoD in 5'-UTR derived ami204 and ami318 and overall down regulation of MRFs in 3'-UTR derived ami204 and ami318 integrated goat myoblasts cells. The 5'- UTR derived ami204 and ami318 showed increased rate of cell proliferation as well as myoblasts fusion in stable goat myoblasts compared to scramble control indicating growth promoting effect of ACVR2B knockdown. The skeletal muscle specific partial knockdown of ACVR2B is unlikely to affect the embryonic survival and it will be interesting to further assess its possible growth promoting effect in adult animal by generating transgenic goat through somatic cell nuclear transfer of goat myoblast cells stably integrated with amiRNAs.ThesisItem Open Access COMPARATIVE EFFICACY OF EGG YOLK AND SOYA BASED EXTENDERS FOR REFRIGERATION (5 °C) AND CRYOPRESERVATION (-196 °C) OF BUFFALO SEMEN(AAU, Anand, 2014) CHAUDHARI, DINESHKUMAR V.; Dhami, A. J.The present investigation was undertaken during the favourable breeding season (November-February) of the year 2013-2014 on six mature Surti buffalo bulls at Central Sperm Station of Department of Gynaecology and Obstetrics of the Veterinary College, AAU, Anand. The study covered evaluation of seminal characteristics in neat semen and then comparative efficacy of egg yolk based standard TFYG (Tris-citric acid-fructoseegg yolk-glycerol) extender and soybean based commercially available extenders (Bioxcell® and Optixcell®, IMV, France) using split-ejaculate technique through various morphological and functional attributes of spermatozoa extended/preserved/ processed in these extenders for refrigeration preservation (at 5°C up to 72 hrs) and cryopreservation (-196°C), including interrelationships of quality sperm parameters of fresh, refrigerated and cryopreserved semen. Immediately after collection, the ejaculates (8 per bull) were evaluated for routine physico-morphological attributes, including motility, viability, morphology (eosinnigrosin), acrosomal integrity (Giemsa stain) and plasma membrane integrity (HOST 150 mOsm/L; an in vitro fertility test) through standard procedures and using phase contrast microscope.ThesisItem Open Access COMPARATIVE APPRAISAL OF PHYSICAL, CHEMICAL, INSTRUMENTAL AND SENSORY EVALUATION METHODS FOR MONITORING OXIDATIVE DETERIORATION OF GHEE(AAU, Anand, 2014) MEHTA, BHAVBHUTI MANOJBHAI; Aparnathi, K. D.Ghee is the most famous traditional dairy product in India, now also gaining popularity in Asian countries as well as other continents like Australia, Europe and America. This fat rich dairy product is highly regarded for typical pleasing flavor. Ghee, being a lipid, prone to oxidation. Lipid oxidation is a degradation process considered to be a major cause of deterioration in the quality of fat products. It imparts rancid and unpleasant flavors to the products and thus decreases their organoleptic value. The reaction of fat oxidation is autocatalytic and occurs in two stages. In primary stage of the fat oxidation reaction between the unsaturated fatty acid and oxygen leads to formation of hydroperoxides and during secondary stage of the reaction the hydroperoxides decomposed into off smelling volatile compounds. This is very complex process that resuh in the formation of several different compounds. Therefore, there is no single method to measure all these compounds, consequently, the use of more than one testing method is recommended to monitor oxidative changes taking place in lipid. Numerous physical, chemical and instrumental methods have been reported to measure oxidative deterioration of oils and fats. The selection of appropriate analytical method is very essential to obtain correct information about oxidative deterioration of product like ghee. Analytical methods most widely reported are weight gain, conjugated dienes, Kreis number, iodine value, free fatty acids content, peroxide value, TBA value, p- Anisidine value, TOTOX value, Carbonyl value and Rancimat. However, no systematic work has been carried out so far for selection of the most appropriate methods to monitor oxidative deterioration of ghee. Therefore, this study was undertaken with a goal to compare performance of selected physical, chemical and instrumental methods to evaluate oxidative deterioration of ghee and to establish correlation between results obtained by these methods with results obtained by sensory evaluation of the ghee for flavor score. The entire work of study was divided as (1) selection of methods used for determination of peroxide value of ghee, (2) evaluation of methods used for monitoring oxidation of ghee on the bases of changes taking place during primary stage of lipid oxidation, (3) examination of methods used for monitoring oxidation of ghee on the bases of changes taking place during secondary stage of lipid oxidation, (4) testing of Rancimat for suitability to study oxidation of ghee, (5) comparison between performance of the method selected from stage of primary oxidation, method selected from secondary stage of oxidation and Rancimat to study oxidative deterioration of ghee and (6) work out the correlation between the results obtained by accelerated storage study and the results obtained from sensory evaluation of ghee for flavor score on storage at normal temperature. In selection of methods for determination of chemical changes taking place during primary stage as well as secondary stage of oxidation in ghee, accelerated storage study was employed. The samples of ghee were prepared by creamy butter method stored at 80°±2°C and monitor for chemical changes using selected methods at regular interval of every 48 hours. The samples of ghee were simultaneously analyzed for changes in flavor score by sensory evaluation using 9 point hedonic scale. For testing the suitability of Rancimat to study the oxidative deterioration of ghee, oxidation curves for the ghee were obtained at 90°C, 100°C, 110°C and 120°C and Arrhenius plots were obtained to predict the shelf life of ghee. Simultaneously ghee samples were also stored at 35°±2°C and monitor for changes in flavor score by sensory evaluation using 9 point hedonic scale at an interval of 10 days. Amongst the BIS, AOAC, AOCS/IUPAC, FOX and IDF methods used to monitor the peroxide formation in ghee, the FOX method was found most consistent. Moreover, the best correlation between changes in flavor score of ghee and peroxide formation was given by FOX method. Amongst the weight gain. Conjugated dienes, Kreis number. Iodine value, free fatty acids content and Peroxide value by FOX method based on chemical changes in the primary stage of the oxidation of ghee, except the FOX method no other method gave significant correlation with changes in flavor score of ghee during storage. Amongst the TBA value, p-Anisidine value, TOTOX value and Carbonyl value based on chemical changes in the secondary stage of the oxidation of ghee, the carbonyl value was found consistent. In addition it gave the highest correlation with changes in flavor score of ghee during storage. The changes in temperatures of Rancimat were highly correlated with induction period of ghee samples and changes in its flavor scores on storage at 80°±2°C. The predicted shelf life of ghee at 80°±2°C obtained from the prediction models prepared using the results of peroxide value by FOX method and carbonyl value of the ghee stored at 80°±2°C and that obtained from Rancimat was almost in corroboration with the actual shelf life of ghee stored at 80°±2°C. The predicted shelf life of ghee at 35°±2°C obtained from the prediction models prepared using the results of peroxide value by FOX method, carbonyl value and flavor score of the ghee stored at 80°±2°C was almost in corroboration with the actual shelf life of ghee stored at 35°±2°C. However, the predicted shelf life of ghee at 35°±2°C obtained from Rancimat was 2.47 times higher than the actual shelf life of ghee stored at 35°±2°C. Therefore, it appeared from the results that use of peroxide value by FOX method, carbonyl value and flavor score of ghee on storage at 80°±2°C are promising for predicting shelf life of ghee at normal temperature, but use of Rancimat does not appear promising to predict the shelf life of ghee on storage at normal temperature.ThesisItem Open Access BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF COLD-INDUCED SWEETENING IN POTATO (SOLANUM TUBEROSUM L.) VARIETIES DURING STORAGE(AAU, Anand, 2014) GALANI, YAMDEU JOSEPH HUBERT; TALATI, J. G.Potato (Solatium tuberosum L.) is the third most important food crop, the most important non-grain food crop and one of the most essential basic vegetable worldwide as well as in Indian subcontinent. After harvest, potatoes are stored in cold storage to provide round the year supply to markets and consumers. But during storage at cold temperatures, many cultivars accumulate free reducing sugars derived from breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose in a metabolic process known as cold-induced sweetening (CIS). Understanding the basis of CIS in potato tubers is of interest not only in basic research on plant adaptation to environmental stress but also in applied research, since high amounts of reducing sugars adversely affect the quality of processed food products. Investigations were carried out to characterize at biochemical and molecular levels the CIS in 11 potato varieties namely DSP 287, DSP 186, Kufri Surya, K. Chipsona-3, K. Sutlej, K. Sadabahar, K. Jyoti, K. Lauvkar, K. Himsona, K. Bahar and K. Badshah stored at 3 different temperatures viz., room temperature (25-32°C), incubator (15°C) and cold storage (4°C). Samples, collected every 15 days intervals for 105 days storage were analyzed for different parameters associated with carbohydrate metabolism on one side, and antioxidant capacity on the other side. Analysis of carbohydrate dynamics showed that low temperature storage negligibly influenced dry matter, starch and maltose contents of tubers, but a significant increase in reducing sugars, total soluble sugars, fructose, glucose, hexoses:sucrose ratio and a decrease of sucrose content were observed at 4°C as compared to room temperature. A strong positive correlation was found between reducing sugars and total soluble sugars, and between fructose and glucose. Additionally, important shrinkage and sprouting of tubers was observed at 15°C, they were less intense at room temperature, and no any shrinkage and sprouting occurred on tubers stored at 4°C. The potato varieties also appeared to be suitable for processing immediately after harvest or short storage at room temperature. The activity of β-amylase was considerably increased by storage at low temperature, and a weak correlation with starch content indicated an important role of other enzymes in starch degradation while absence of maltose accumulation with increased β-amylase activity implied a possible significant activity of maltase in potato tubers. Acid invertase activity drastically rose at low temperature and strongly paralleled reducing sugars, glucose, fructose and hexoses:sucrose ratio. Moreover, as acid invertase activity increased, sucrose content decreased, indicating the essential role of acid invertase in development of CIS. The above findings allowed to group the 11 potato varieties into low to high sugar-forming groups and thereby select K. Jyoti as CIS-tolerant and K. Badshah as CIS-susceptible. Banding pattern of both native and denaturated proteins of potato tubers could not clearly discriminate the varieties. Zymograms of p-amylase revealed differentially induced bands at low storage temperatures, which might justify the observed increase of enzyme activity. Screening of the 11 potato varieties with 10 SSR primers detected a total of 42 alleles arranged in 44 different configurations, among which 37 alleles (88%) were polymorphic. The polymorphic information content value of the SSR locus ranged from 0.473 to 0.787 thus indicating a high utility of these markers for study of genetic diversity in potato. The dendrogram derived from Dice's similarity coefficients among the 11 varieties could partially but efficiently differentiate close parents and sugar-forming groups. Differential gene expression analysis showed that during storage expression of vacuolar acid invertase gene StvacINV1 and p-amylase gene BAMl increased at low temperature and their transcripts were more expressed in the CIS-tolerant variety than the CIS-sensitive. Expression of invertase inhibitor gene INH2a however was higher in the CIS-tolerant variety than the CIS-sensitive. Correlating StvacINVl and INH2a expressions with reducing sugar content and acid invertase activity established that post-translational regulation of acid invertase by the invertase inhibitor protein could be an important component of resistance to CIS. Besides, correlation between BAM1 expression and (β-amylase activity affirmed the hypothesis of several enzymes and pathways involved in starch degradation during cold storage of potato. Analysis of antioxidant capacity parameters revealed that low temperature storage greatly influenced vitamin C content as well as the phenolic content. During storage, both parameters initially increased, then a fluctuated decline was observed but until the last day of observation, they remained above the initial level. Phenolic acids profiling by UPLC identified 12 compounds among which the most abundant was chlorogenic acid followed by gallic acid, sinapic acid and ellagic acid which is reported for the first time, while trans-cinnamic acid was the lowest. Except paracoumaric acid which decreased at 4°C, all the phenolic acids increased with storage among which sinapic acid and feruhc acid appeared to be most enhanced. Correlation analysis showed that gallic acid, caffeic acid. chlorogenic acid and protocatechuic acid significantly contributed to total phenolic content. Evaluation of antioxidant activity showed a close relationship between DPPH and ABTS methods. Antioxidant activity estimated by both the methods increased up to 60 days storage then at 90 days, they dropped to a level comparable or lower than the original value, irrespective of the storage temperature. Correlation study revealed that chlorogenic acid, gallic acid and ferulic acid mostly contributed to antioxidant activity. Activity of the antioxidant enzymes superoxide dismutase and ascorbate peroxidase both increased initially but then decreased to values lower than the initial level and were not influenced by storage temperature. Correlation with antioxidant activity indicated that the enhancement of reactive oxygen scavenging species in tubers could result mainly from ascorbate peroxidase activity. Isoforms of the two enzymes showed interesting polymorphism and changes in bands intensity as well as differential induction or suppression of bands during storage. However, isozymes of ascorbate peroxidase showed higher similarity and better discrimination of the varieties. Although a clear relationship between CIS and antioxidant capacity was not established, nevertheless it appeared that low sugar-forming varieties K. Jyoti, K. Himsona and K. Surya were also having high antioxidant capacity whereas K. Chipsona-3 and K. Bahar both high sugarforming had low antioxidant capacity. Hence, it is not unreasonable to suggest that antioxidant capacity of potato tubers should be taken into account in development of CIS-resistant varieties. Nonetheless, additional evidences are needed to confirm this suggestion as well as there is an urgent need to develop new varieties capable to cope with this cold stress.ThesisItem Open Access PERSISTENCE OF PROFENOPHOS AND TRIAZOPHOS IN SANDY LOAM SOIL UNDER LABORATORY CONDITIONS AND THEIR DISSIPATION IN/ON BRINJAL AND TOMATO FRUITS UNDER SUPERVISED FIELD TRIALS(AAU, Anand, 2014) RANA, GAJENDRA KUMAR; Shah, P. G.Vegetables are important ingredient of our food having a high nutritional value. Brinjal and tomato are important solanaceous crops of India, cultivated throughout the country and constitute an important part of human diet. The vegetable yield in India is considerably low because of several factors, the most important being the damage caused by various insect pests like fruit and shoot borer, jassids, aphids, leafminer etc. In order to protect the crops from such pest damage, former predominantly rely on chemical pesticides. Use of organochlorine (OC) group of pesticides is banned in agriculture primarily due to their persistence in the environment. However, use of organophosphate group of pesticides is on rise mainly due to their easy availability and quick degradation in the ecosystem. Of these, profenophos and triazophos have been found quite effective for the management of these pests. Triazophos (O, O-diethyl-O-1-phenyl-1H-1, 2, 4-triazol-3-yl phosphorothioate) is a contact and stomach insecticide. The chemical primarily controls sucking and chewing insects in many crops. In spite of being non-systemic, triazophos can penetrate deeply in the plant tissues due to its translaminar properties and can effectively control leaf miner. Triazophos is broken down in the environment and does not bioaccumulate, unlike certain organochlorine insecticides. The insecticide is dangerous to bees and harmful to fish, livestock, birds and other animals. Profenophos [O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorohioate) is an insecticide and acaricide belonging to organophosphate group. It is an insecticide used on a wide variety of crops to control pests mainly Lepidoptera and mites. It was developed for those insect strains which developed resistant to other organophosphorus pesticides. It is selectively more toxic to insects as compared to mammals. For better production and high value, farmers are using a large amount of insecticides during the entire period of cultivation, even at fruiting stage. Quite often farmers also ignore the recommended dose and suggested waiting period between the harvest and last spray. Thus, injudicious use of pesticides could pose serious risk to the consumers besides contaminating the fields. Monitoring studies carried out worldwide have reported the residues of pesticide in fruits and vegetables. Though there is no label claim for profenophos and triazophos on tomato and brinjal in India, monitoring of pesticides residues in fruits and vegetable has revealed presence of these insecticide in/on brinjal and tomato fruits. The dissipation of these insecticides varies with their physical and chemical, dosage applied, number of applications, interval between applications, crop variety, agro climatic conditions, etc. Hence, a study entitled " Persistence of profenophos and triazophos in sandy loam soil under laboratory conditions and their dissipation in/on brinjal and tomato fruits under supervised field trials" was proposed to know the dissipation and persistence of profenophos and triazophos under laboratory as well as field conditions with the following objectives. 1. To study the recovery of profenophos and triazophos in sandy loam soil 2. To study the persistence of profenophos and triazophos in sandy loam soil under laboratory conditions 3. To study the recovery of profenophos and triazophos in tomato and brinjal fruits 4. To study the dissipation of profenophos and triazophos in tomato and brinjal fruits under field conditions 5. To study the effect of washing and cooking in the reduction of profenophos and triazophos residues in brinjal and tomato fruits A field experiment was conducted during Rahi 2012-13 at Main Vegetable Research Station, AAU, Anand (Gujarat), to study the dissipation of profenophos and triazophos in both brinjal and tomato fruits. The experiment was carried out in Randomized Block Design (RBD) with three replications along with a control plot. In brinjal and tomato, two sprays of profenophos and triazophos insecticides were applied during fruiting stage @ 500 g a.i. ha-1 at an interval of 10 days. An untreated control was maintained for comparison. Immediately after the last application (i.e. one hour after spray) fruit samples were drawn for 0 day. The subsequent sampling was carried out 1st, 3rd, 5th, 7th and 10th day after second application. A laboratory trial was also carried out to determine the persistence of profenophos and triazophos in sandy loam soils. A representative 20 g (dry weight equivalent) sample of sandy loam soil in 50 mL polypropylene tube was taken and adjusted to 20 % moisture content by addition of distilled water. Solutions of the insecticides (profenophos and triazophos) in acetone were applied drop wise to the surface of the soil in each tube to obtained concentration 10 mg a.i. kg-1 The samples were drawn at 0 (1hr after application), 1, 3, 5, 7, 10, 15, 20, 25, 30, 40, 50 and 60 days after application. Residues of profenophos and triazophos were estimated by a validated GC-PFPD method.ThesisItem Open Access STANDARDIZATION AND CLINICAL USE OF DIODE LASER IN DOGS(AAU, Anand, 2014) TARWARE, VARSHA; PARIKH, P. V.LASER is the acronym for Light Amplification by Stimulated Emission of Radiation. The diode LASER is one of the most widely used medical lasers in the world delivering an intense beam of infrared light with 635-980 nm wavelengths. In the present study, selected clinical cases were allotted to two groups. In Group 1, six animals (dogs) were subjected to celiotomy with diode LASER technique and in Group 2, twelve dogs with small tumors (<3 cm) were subjected to diode LASER ablation. The dogs were prepared aseptically for the surgeries. After premedication with Inj. Atropine (@ 0.02 mg/kg body weight), dogs were anaesthetized with Inj. Ketamine HCl (@ 10 mg/kg body weight) and Inj. Diazepam (@ 0.5 mg/kg body weight) IV. Full thickness skin was incised using StarLas 250 diode LASER with 0.2 mm ceramic tip and settings of 7-9 watt (in continuous wave mode) and up to 25 watt in pulsed mode. The laser tip (hand piece) was kept in contact mode. Sterile saline solution moistened gauze sponges were used to remove carbonized debris which facilitated cutting of tissue with the laser and avoiding thermal injuries to under lying structures. During LASER surgery, surgical blood loss was estimated, and postoperative complications, surgical outcome and postoperative clinical appearance were recorded after 15 to 30 days. In the present study, power settings used for warts / tumors (3-14 WCW mode and up to 25 W- pulsed mode) and celiotomy (7-9 W - CW mode and up to 25W - pulsed mode) were optimum. Diode LASER was also used for surgeries viz. aural hematomas, oral tumors, epilation of eyelid hairs and entropion. The average blood loss during surgery was ~ 1 ml. After 15-30 days of LASER surgery, there was desired wound healing with cosmetic appearance of scar and overall less wound complications. Diode LASER is an effective excisional and dissectional tool which provides significant cutting and coagulation ability in contact mode and thus has superior hemostatic abilities. Based on observations of the present study, it can be said that diode LASER can be a part of surgical armamentarium of small animal practitioners and its judicious use will improve surgical results with cosmetic appearance of the wound.ThesisItem Open Access PATHO-EPIDEMIOLOGICAL STUDY ON GENOTYPE-XIII NEWCASTLE DISEASE VIRUS INFECTION IN COMMERCIAL CHICKEN(AAU, Anand, 2014) KHORAJIYA, JAYNUDIN H.; JOSHI, B. P.The present research work was carried out to know Patho-epidemiological study on Genotype-XIII Newcastle disease virus infection in commercial layer and broiler chicks in around Anand, Gujarat. The Genotype-XIII Newcastle disease virus was confirmed by F gene sequence and whole genome sequence at Department of Microbiology and Animal biotechnology, Veterinary College, Anand. The study comprised of patho epidemiology of Newcastle disease by information collected from different broiler and layer farms suffered from the disease in relation to incidence pattern and mortality, duration of mortality, susceptible age, loss due to production performance, clinical signs, gross and histopathological lesions of visceral organs as well as to isolate and identify pathotype of virus by HA, HI and ICPI test. During the study mortality due to Newcastle disease was recorded in 13 layer and 10 broiler flocks inspite of routine vaccination which usually contain genotype-II strain of virus. The mortality was observed above 50 percent with an average of 21.21 percent in layer flocks and to the tune of 80 percent with an average of 28.11 percent in broiler flocks. The susceptible age of the disease was found to be 23 to 34 days among broiler and 6-14 weeks among layer flocks. The duration of mortality observed was 14 days in broiler flocks and 23 days among layer flocks. The disease resulted in significant reduction in body weight upto 22.80% in layer flocks and 29.06 % in broiler flocks in comparison to standard normal body weight. There was significant reduction in feed intake upto 35.52% in broiler flocks and 46.91 percent in layer flocks. The two affected egg laying flocks showed drop in egg production to the tune of 20 to 30 percent. Majority of the outbreaks appeared during extreme hot months of may and June in broiler flocks and april to June in layer flocks. The major clinical signs presented by the affected flocks were listlessness, increased respiration, greenish diarrhoea with soiled feathers of vent, dehydration, loss in body weight, conjunctivitis, prostration and increasing mortality. Greenish diarrhea was frequently seen in birds that survived early in infection. Mortality continued for 2-3 weeks and reduced with appearance of torticollis. Gross lesions were characterized by emaciation and dehydration of carcass with deep congestion of breast musculature, multifocal to diffuse haemorrhages around proventricular glands, necrotic (diptheretic) haemorrhagic ulcers throughout the intestine, disseminated multiple foci of necrosis and pin-point haemorrhages in spleen parenchyma especially in layer birds. In few of the broiler flocks kidneys appeared pale and enlarged with pin point haemorrhages. Severe congestion of trachea and lungs was prominent feature in majority of broiler as well as layer chicks. The microscopic lesions were mainly of the nature of focal to diffuse haemorrhages and diffuse infiltration of mononuclear cells in proventricular mucosal and glandular regions; focal to diffuse haemorrhages, necrosis and sloughing of epithelial cells and moderate to severe infiltration of mononuclear cells in the intestine; multifocal areas of lymphoid necrosis and haemorrhages in caecal tonsils, spleen and bursa of fabricius; and perivascular infiltration of lymphocytes, neuronal degeneration and focal areas of gliosis in brain parenchyma. Pooled tissue samples (trachea, lung, liver, spleen, proventriculus, caecal tonsils and intestine) collected aseptically during postmortem examination from all the 23 flocks of broiler and layer farms were homogenized and tissue suspensions were inoculated into the allantoic cavity of embryonated SPF eggs of 9-11 days incubation. Eggs were further incubated until death or for upto 72 hrs and allantoic fluid was collected after overnight chilling at 4°C and used for further HA, HI test and ICPI test. All the 23 allantoic fluids from field samples along with F and R2B vaccine sample were found positive for HA activity, which was further confirmed by HI using known NDV serum. The values of intracerebral pathogenicity index (ICPI) carried out for assessment of virulence of Newcastle disease virus in day old SPF chicks for the filed samples were 2.0 and indicative of velogenic nature of the filed NDV strain. The clinical signs, mortality pattern, gross and microscopic lesions, haemagglutination (HA), haemagglutination inhibition (HI) activity and ICPI (Intracerebral Pathogenicity Index) observed in filed outbreaks of Newcastle disease both in layer and broiler flocks were indicative of very virulent Newcastle disease. Further isolation of genotype-XIII NDV from the filed outbreaks and ICPI score of 2.0 confirmed that the present outbreaks were due to genotype-XIII Newcastle disease virus which was velogenic in nature. The study indicated that presently available live and attenuated vaccines which include genotype-II NDV have failed in protecting the flocks against genotype-XIII and resulted in outbreaks with mortality above 50 percent in layer flocks and upto 80 percent in broiler flocks.ThesisItem Open Access STUDIES ON ELECTRORETINOGRAPHY USING RETlport ERG SYSTEM IN CANINES(AAU, Anand, 2014) KELAWALA, DIVYESH NARESHBHAI; PATIL, D. B.In the era of information technology, the demand from the pet owners for better diagnosis of retinal disorders is increasing, hence it was envisaged to gain step by step technical skill and standardize the technique of electroretinographic study in dogs. The present study was conducted in 20 dogs, of which ten had no vision abnormality (Group A) and the rest with vision abnormalities (Group B) viz., cataract, retinal detachment, vitreous degeneration, progressive retinal atrophy, retinal degeneration, etc. The ERGs of normal and affected dogs were obtained with special reference to standardize RETIport ERG system and to establish ERG system-specific limits of normality in order to obtain a correct diagnosis. Prior to ERG, all dogs were subjected to detailed ophthalmic examination. Scotopic and photopic ERGs were obtained under general anaesthesia following the protocols of ISCEV. ERG's were interpreted using amplitude and implicit times of and b-waves. The range of implicit time and amplitude of the scotopic b-wave for Group A dogs was 26-49 (median: 37.5) msec and 1.3-505 (median: 253.15) μV, respectively. Range of the implicit time and amplitude for the scotopic a-wave was 8- 21 (median: 11) msec and 23.2-371 (median: 85.7) μV, respectively. The range of implicit time and amplitude of the photopic b-wave for Group A dogs was 16-45 (median: 23.5) msec and 1.3-966 (median: 65.5) μV, respectively. Range of the implicit time and amplitude for the photopic a-wave was 5-19 (median: 12) msec and 2.92-502 (median: 49.7) μV respectively. The Mean+SE of b/a ratio for Group A dogs was 2.33+0.73. The normal range for each breed should be established for ERG to become a more valuable examination tool and the parameters obtained in this study i.e. b/a ratio (2.33+0.73) for dogs with normal vision can be used as normal ERG reference ranges for Spitz dogs. The causative factor for vision abnormality was PRA in three dogs (30%) followed by cataract (n=2; 20%), retinal degeneration (n=2; 20%) and one case each of PRA with cataract, glaucoma and CKD. Dogs with PRA displayed non-recordable extinguished (flatline) ERG response, which confirmed a loss of retinal fianction of the eyes. In a dog with CKD induced retinal detachment, there was rapid reduction in the amplitude of b-wave, but the b/a ratio was within the normal range (2.02+0.38) which indicated that the detached retina is electrically active. The scotopic b/a ratio for the eye with glaucoma was lower and photopic b/a ratio was higher than the normal ranges. In dog with sudden loss of vision, the scotopic b/a ratio of right eye was higher than normal range, while for left eye and photopic b/a ratio for both eyes was lower than normal, propbably indicative of SARD. Non-detectable scotopic rod responses and scotopic maximal response were found in flash ERG of a dog with congenital retinal degeneration. In eyes with mature and hypermature cataracts, there was decrease in the b-wave amplitude and mean peak to peak amplitudes smaller with b/a ratio higher than the normal range. Thus, ERG is useful adjunct test for the diagnosis of retinal dystrophies and for pre-operative evaluation of retinal functions in conjunction with cataract surgery.