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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 DETECTION OF POLYMORPHISM IN BOVINE PREIMPLANTATION ACTIVE GENES AND THEIR ASSOCIATION WITH SEMEN QUALITY(AAU, Anand, 2008) AHIR, VIRAL B.; Panchal, M. T.Embryo development in mammals is marked by distinctive biological processes that occur during the preimplantation and early post implantation periods. Preimplantation development encompasses the period from fertilization to implantation, which occurs in different times in various species and it is marked by a number of critical events. This development is a mammalian-specific event, and is vital for successful implantation and pregnancy. Bovine preimplantation embryo development is under constant control of genes activated from either maternal or embryonic genome. Large-scale association studies by genotyping many single nucleotide polymorphisms (SNPs), in individuals with well characterized phenotypes, are considered as promising methods to identify the cause of many complex traits. The present study was undertaken to study the polymorphism of bovine preimplantation active genes loci by PCR-RFLP and PCR-SSCP techniques and their association with various semen quality traits in Murrah buffalo bulls belonging to ARDA, Ode during January to June 2008. The mean and standard error of mean (SEM) for various semen quality parameters, viz., volume (ml), concentration (106/ml), motility (%), motility after thawing(%) and live and dead count (%) was found to be 3.35+0.27, 1511.07+112.25, 73.54+1.05, 54.39+0.66 and 85.44+0.47, respectively. Semen DNA was extracted from 41 Murrah buffalo bulls by Proteinase K method as per standard protocol. Bovine COX-2, CD9, DSC2, AKRIBI and CDHl genes specific primers (COX-2 F: 5'-TGA TCT ACC CGC CTC ATG TT-3' and COX-2 R: 5'-CCC TTT GCC TGG TGA ATG-3'); (CD9 F: 5'-GAG GCA AAA CTC CAA AAC CA-3' and CD9 R: 5'-CTC CAC TGT CGT TTG TCG TG -3'); (DSC2 F: 5'-AAA GTG CAA GAC ATG GAT GG-3' and DSC2 R: 5'-CCT TCA TTG GTT TGG GAA TC-3'); (AKRIBI F: 5'-ACC AGG GCT TAC CTG GAA GT-3' and AKRIBI R: 5'-GGT CAA TGG GCC TTA GGA TT-3') and (CDHl F: 5'-CGC ACA ACA AAA TGT TCA CC-3' and CDHl R: 5'-GGC CTC AAA TCT CCA GAC AA-3') were used to amplify bovine preimplantation active genes loci. PCR was carried out in 25 µl volume for 35 cycles of denaturation at 94°C, annealing at appropriate temperature (COX-2 locus at 52°C, DSC2 and CDHl loci at 51°C, AKRIBI locus at 58°C) and extension at 72°C. Initial denaturation was carried out at 94°C for 5 minutes, while the final extension was performed at 72°C for 5 minutes. For the CD9 locus "Touch down PCR" was performed to avoid spurious priming during PCR amplification. Amplified products were electrophoresed on 2% Agarose gel at 80 V for for 60 minute. For RFLP analysis Amplified PCR product of COX-2, CD9, DSC2 and AKRIBI loci were digested with Alu I, Dra I, Rsa I and Nde /restriction enzymes respectively, by incubating them at 3 7°C for 14-16 hours except for Rsa I which was incubated at 37°C for 12 minute and electrophoresed on 2% Agarose gel at 80 V for 60-90 minutes to reveal the restriction pattern. Monomorphic pattern was observed for the COX-2, CD9 and DSC2 loci and only TT, TT and AA genotypes, respectively, were found in all Murrah buffalo bulls at these loci. The allelic frequencies of T, T and A alleles were 1.00, with absence of A, A, and G alleles, respectively. For AKRIBI locus, 18 samples were found to be homozygous AA and 23 samples were heterozygous AG, with allelic frequency of 0.725 and 0.275 for A and G alleles, respectively. For SSCP analysis, PCR products of CDHl locus were denatured and electrophoresed on 6% non-denaturing PAGE for 4-5 hours at constant 5W. Analysis revealed four different banding patterns with frequencies for pattern 1, pattern 2, pattern 3 and pattern 4 to be 0.463, 0.146, 0.292 and 0.097, respectively. Since, loci COX-2, CD9 and DSC2 were found to be monomorphic, h was not possible to correlate them with the semen quality traits. Loci AKRIBI and CDHl were found to be polymorphic but, statistical analysis revealed no significant association (P>0.05) of this loci with any semen quality parameters.ThesisItem Open Access ISOLATION OF PPR VIRUS, DETECTION BY ELISA AND DIFFERENT GENE BASED RT-PCR(AAU, Anand, 2008) CHOUDHARY, POOJA; Jhala, M. K.Peste des petits ruminants (PPR) is a severe viral disease of goats and sheep with high morbidity and sometimes high mortality characterized by fever, erosive stomatitis, conjunctivitis, gastroenteritis and pneumonia. PPR is caused by Peste des petits ruminants virus (PPRY), a Paramyxovirus of the Morbillivirus genus. The disease causes severe economic losses to small ruminant husbandry and is presently considered as one of the major threats to about 200 million small ruminant population of the country. The present study was aimed to detect PPRV in clinical samples using s-ELISA and to derive estimates of overall, locationwise and specieswise incidence of PPRV. The study also involved the isolation of PPR virus from clinical samples and assessment of F, N and H gene targets for detection of PPRV by RT-PCR from the clinical and cell culture samples. A total of 98 clinical samples comprising of 48 tissues, 12 blood and 38 serum samples from 79 animals including 26 sheep and 53 goats suspected of PPRV infection, were collected from three different districts of Gujarat (Rajkot, Bhavnagar and Gandhinagar) and tested for PPRV antigen by s-ELISA (PPR s-ELISA kit, developed by Division of Virology, IVRI, Mukteshwar), of which 75 animals were found positive yielding an overall incidence rate of 94.90 per cent. Out of 10 animals (goat) suspected of PPR, six were positive (60%) by s-ELISA in Rajkot district. All the 32 and 37 animals (sheep and goats) showing clinical signs suggestive of PPR from Bhavnagar and Gandhinagar districts were found positive by s-ELISA yielding 100 %'incidence at both these locations. Sheep was found to be more susceptible to infection yielding a positivity rate of 100 per cent than goats (92.4%). The PPR antigen could be detected marginally more in tissue samples (95.83%) than in blood (83.33%) and serum samples (89.47%) by s-ELISA. A total of 23 samples including 15 cell culture samples, six tissue samples, one whole blood and reference vaccine virus (Sungri isolate) were processed for RNA extraction using TRI Reagent®. RNA samples showing acceptable purity and concentration were reverse transcribed with random hexamers to generate cDNA templates, which were subjected to PCR amplification using F, N and H gene based primers. Reference vaccine virus as well as 10, 11 and 8 out of 15 cell culture samples of the three isolates produced approximately 372 bp amplicon with F1 / F2, 351 bp amplicon with NP3 / NP4 and 713 bp amplicon with H1 / H2 primers respectively. A total of 23 samples were tested by both s-ELISA and RT-PCR, including reference vaccine virus, seven field samples (six tissues, one whole blood) and 15 cell culture samples. Out of the total samples tested, PPRV could be detected in reference vaccine virus and 19 samples by s-ELISA and 15, 18 and 11 samples including reference vaccine virus by F, N and H gene based RT-PCR respectively. None of the sample positive in RT-PCR was negative by s-ELISA. Relative to s-ELISA, sensitivity of the RT-PCR for F, N and H gene was 78.94, 94.70 and 57.89 respectively and specificity for all the three was 100 per cent. Three representative samples comprising of one pooled tissue sample, one whole blood sample and one serum sample were inoculated in Vero cells for five viral passages to isolate the field PPR virus. All the three samples inoculated showed similar CPE. During first passage, changes associated with rounding of cells and isolated initiation foci were observed. During second and third passages, CPE characterized by ballooning of cells and later aggregation of cells followed by formation of fusion mass and syncytia were recorded. Cell lysis was also observed in few cases. During fourth and fifth passages, the above mentioned CPE increased in intensity with more number of cells showing fusion mass and detachment. The monolayer infected with sterile PBS (negative control) did not show such changes. The isolation of PPRV in cell culture was confirmed by s-ELISA after second passage for all the three samples and after P3, P2 and P2 passage by F, N and H gene based RT-PCR.ThesisItem Open Access MOLECULAR CHARACTERIZATION AND DIVERSITY OF RUMEN PROTOZOA IN BUFFALO(AAU, Anand, 2008) NISHA; Pandya, P. R.Rumen harbors diverse types of microbes mainly bacteria followed by protozoa, fungi and yeast. Bacterial population is as high as 10 to power 10 and protozoa population is 10 to power 5 to 10 to power 6 per ml of fluid, found in the rumen and has a profound effect on nutritional and physiological processes in the host. The ability and characteristic of rumen microbes differs. Some are good at digesting concentrate while others at roughage. The study of rumen ecology involves their relationship with each other and the host. Much of the pioneering studies on the rumen microbiota are based on microscopic examination and anaerobic culture techniques. But the polymorphic nature and the difficulty of cultivating the microbes have hampered effective assessment of ruminal ecology by these methods. Newer molecular approaches are available to identify and characterize the eukaryotes and prokaryotes that are based on detecting highly conserved gene regions-16S rRNA and 18S rRNA, respectively for bacteria and protozoa. India possesses more than 50 per cent of world buffalo population. The present study was aimed to determine genetic characterization of rumen protozoa in buffalo and their phylogenetic classification, based on sequence comparison of 18S rRNA gene of rumen protozoa. Three adult buffaloes were maintained on standard dietary regimen as per ICAR (1998) feeding standards, composed of green and dry roughage and compound concentrate mixture, continuously for three weeks. Samples of Rumen liquor (about 500 ml) were collected from three buffaloes at 2, 4 and 6 hrs. after feeding by a suction pump using a flexible stomach tube. Samples were filtered through four layers of cheesecloth. The strained rumen liquor was used for the study. The protozoal DNA was isolated following enzyme-chemical lysis method. The DNA stock samples were quantified using Nano-drop spectrophotometer at 260 and 280 nm using the convention that one absorbance unit at 260 nm wavelength equals 50 µg DNA per ml. The Ultra violet absorbance was checked at 260 and 280 nm for determination of DNA concentration and purity. Quality and purity of DNA were also checked by agarose gel electrophoresis. Protozoa specific primers viz. Euk-82F 5' AAA CTG CGA ATG OCT C -3' and Medlin Be 5' TGA TCC TTC TGC AGG TTC ACC TAG -3' targeting 18S rRNA gene were used for amplification of DNA. The amplified product was visualized as a single compact band of expected size under UV light and revealed amplicons of 1346 bp size when documented by gel documentation system. The PCR products after purification were ligated in pDrive vector followed by transformation into competent cells (DH5-a strain) of E.coli. One hundred fifty white recombinant colonies were obtained out of which 124 were randomly selected, revived on another plates and screened for expected insert by colony PCR. Recombinant colonies were inoculated in Luria Broth for 16-18 hrs. Plasmid extraction from overnight culture was carried out by alkaline lysis method. The concentration of the plasmid DNA was determined and was subjected to automated DNA sequencing on ABI PRISM® 310 Genetic Analyzer (Applied Biosystems, USA). Sequencing was carried out using BigDye® Terminator v3.1 Cycle sequencing kit. Sequences obtained were in the range of 250 to 850 base pairs. Sequences after cleaning (removal of primer and vector sequence) were searched against BLASTn database to find similarity matches. Analysis of the 18S rRNA sequences of protozoal species from ruminal fluid revealed predominance of family Isotrichidae {Dasytricha and Isotricha) and Ophryoscolecidae. Phylogenetic analysis was performed using DNADIST and DNAPARS (Parsimony method) program of PHYLIP package (Felsenstein, 1989). Phylogenetic tree was constructed by neighbor-joining method (Saito and Nei, 1987). Out of 124 clones, 21.7% showed similarity with Ophryoscolex spp., 7.2% showed similarity with Cycloposthium spp., 18.5% with Trichostomatidae, 1.6% with Haptorians and about 51% clones remained as unclassified protozoa which are supposed to be new. In DNADIST analysis, two clones (1.6%) from 124 sequenced clones fall in Haptorian group. The AVCRPN62 clone showed 99% similarity with the Enchelyodon spp. while AVCRPN86 showed 99 % similarity with Arcuospathidium spp. In DNAPARS analysis, AVCRPN62 clone showed 95% similarity with Arcuospathidium spp. and was clustered separately in subcluster II of Cluster III. Twenty seven clones (21.7%) clustered separately with Ophryoscolex spp. (21 clones) and Spathidium spp. (6 clones). Members of this group showed 82% similarity in DNADIST analysis. No clones showed similarity with this group in DNAPARS analysis. Nine clones (7.2%) were showing 94% similarity with Cycloposthium spp. which belonged to Cycloposthiidae family and were grouped in Cluster IV in DNADIST analysis, while 24 clones were showing similarity with Cycloposthium spp. and were grouped in subcluster 1 in Cluster III in DNAPARS analysis. Twenty three clones (18.5%) out of 124 clones were showing similarity with Trichostomatidae and were grouped separately in Cluster V in DNADIST analysis, while 36 clones were showing similarity with Trichostomatidae and were grouped in subcluster IV of Cluster III in DNAPARS analysis. Members of Cluster V showed 77% similarity in DNADIST analysis. This cluster was again divided in four sub clusters:Australian clade, Entodiniomorphs, Dasytricha spp., Isotricha spp. Sixty three clones (51%) clustered separately which were showing no significant similarity with cultured as well as uncultured representatives. Clones in this group can be considered as novel clones. Taxonomic classification of these clones showed that the maximum no of clones show similarity to Isotricha spp., Dasytricha spp. and uncuhured rumen protozoa. All the sequences were submitted to Genbank and are available with the accession numbers EU796091- EU796211 in EMBL, GenBank and DDBJ Nucleotide Sequence Databases.ThesisItem Open Access PARENTAGE VERIFICATION IN FIELD PROGENY TESTING PROGRAMME OF MEHSANA BUFFALO BY MICROSATELLITE ANALYSIS(AAU, Anand, 2008) JAKHESARA, SUBHASH J.; Solanki, J. V.The knowledge of correct parentage is a prerequisite in breeding programmes. The identification of proven sires has been of utmost importance in animal improvement programmes. Failure to record correct parentage can cause bias in sire evaluation, by introducing errors in estimates of heritabilities and breeding values. Microsatellites are preferred molecular markers for individual identification and parentage verification. Present study aimed at verifying parentage in daughters covered under progeny testing program operated by Dudhsagar Research and Development Association (DURDA), Mehsana. A total number of 212 Mehsana buffalo samples including 100 daughters for parentage testing, 12 sires and 100 dams were genotyped. Multiplex primer panel containing set of eleven fluorescent labeled microsatellite markers (CSSM61, ILSTS29, ILSTS17, ILSTS28, CSSM43, CSSM57, CSSM22, ILSTS61, CSSM8, ETH152, ILSTSl 1) was developed after screening of 22 microsatellites selected fi-om available list of 25 microsatellites suggested by the National Bureau of Animal Genetic Resources (NBAGR) and 25 cattle specific microsatellites. Primers were selected after their successful amplification individually and in multiplex panel. Multiplex panel was designed in a way that amplification of any primer does not overiap with amplification of other primer with the same dye. Multiplex panel was standardized and optimized for primer concentration and annealing temperature. Eleven microsatellites were amplified in single multiplex for automated fluorescence genotyping to verify parentage in Mehsana buffalo breeds. The number of alleles varied from 5 for marker ILSTSll to 16 for marker ILSTS61. The heterozygosity of the 11 different markers ranged between 0.281 for marker CSSM43 and 0.821 for marker ILSTS61. Calculated PIC values ranged from 0.80 highest for marker CSSM61 to 0.60 lowest for marker ILSTS29. Mean number of alleles per locus (k) was 9.91, Mean observed heterozygosity (HObs) was 0.675, Mean expected heterozygosity (HExp) was 0.762, Mean PIC observed was 0.730. Exclusion probability was highest for marker ILSTS61 and CSSM 61(0.49) and lowest for marker ILSTS29 and CSSM57 (0.25). The combined exclusion probability of all 10 markers was 0.993. Combined exclusion probability using five most polymorphic markers was 95.5 % and for all ten markers was 99.3 %. Paternity of 19 of 100 daughters (19%) was excluded because putative paternal alleles were not present in the progeny for at least two locus. Parentage was assigned to 95 daughters with strict confidence using Cervus 3.0 and five daughters were declared unassigned. Parentage verification also performed manually to check the results obtained by Cervus and they were in accordance. The developed panel of 10 microsatellites in a single multiplex constituted a fast, robust, reliable and economic tool to verify the parentage and assign the putative sire to daughters under progeny testing with very high accuracy. This can be used in routine parentage testing.ThesisItem Open Access “Detection of Polymorphism in Bovine Preimplantation Active Genes and Their Association with Semen Quality(Anand Agricultural University, Anand, 2008) Viral B. Ahir; Dr. M.T. PanchalEmbryo development in mammals is marked by distinctive biological processes that occur during the preimplantation and early post implantation periods. Preimplantation development encompasses the period from fertilization to implantation, which occurs in different times in various species and it is marked by a number of critical events. This development is a mammalian-specific event, and is vital for successful implantation and pregnancy. Bovine preimplantation embryo development is under constant control of genes activated from either maternal or embryonic genome. Large-scale association studies by genotyping many single nucleotide polymorphisms (SNPs),ThesisItem Open Access Identification of Polymorphism in Bovine CYP1A1 gene segment by SSCP and DNA Sequencing(Anand Agricultural University, Anand, 2008) Rakesh kumar Chaturbhai Parikh; Dr. S. K. BhavsarThe most important drug metabolizing enzyme family, Cytochrome P450 (CYP) is one of the conserved entities among species. The cytochrome P450 enzyme system is responsible for the metabolism and clearance of a wide array of drugs, toxins and endogenous substrates. The family is involved in metabolism of 70 – 80% of drugs, nutrients, endogenous substances and environmental toxins. Studies concerning Cytochrome P450 polymorphism, expression and regulation have received very little attention in veterinary species (e.g. cattle, swine, poultry), compared to man and laboratory animals. These species are often exposed to drugs, pesticides or pollutants potentially harmfulThesisItem Open Access “Molecular characterization and Diversity of Rumen Protozoa in Buffalo(Anand Agricultural University, Anand, 2008) NISHA; Dr. P.R. PandyaRumen harbors diverse types of microbes mainly bacteria followed by protozoa, fungi and yeast. Bacterial population is as high as 1010 and protozoa population is 105 to 106 per ml of fluid, found in the rumen and has a profound effect on nutritional and physiological processes in the host. The ability and characteristic of rumen microbes differs. Some are good at digesting concentrate while others at roughage. The study of rumen ecology involves their relationship with each other and the host.ThesisItem Open Access FULL LENGTH cDNA SYNTHESIS OF DIFFERENTIALLY DISPLAYED ESTs DURING LACTATION IN INDIAN BUFFALO (Bubalus bubalis(Anand Agricultural University, Anand, 2008) AJAI KUMAR TRIPATHI; Dr. C. G. JoshiTechnological developments in a variety of scientific and engineering disciplines will be needed to support the growing world population, which is expected to double in the next 40 years. It has been estimated that the supply of food required to adequately meet human nutritional needs over the next 40 years is quantitatively equal to the amount of food previously produced throughout the history of humankind. To meet this need, it will be essential that scientists continue to develop new technologies that increase productivity. A key challenge to be met by biotechnologist, genetician and dairy scientists is to understand which genes control the composition of milk, how these genes are regulated and how they might be manipulated to enhance both, the manufacturing and health properties of dairy productsThesisItem Open Access MOLECULAR ANALYSIS OF STAT5A AND PPARGC1A LOCI IN MEHSANA BUFFALOES (Bubalus bubalis(Anand Agricultural University, 2008) PATIL AJAY VITTHAL; Dr. P. H. VataliyaIndia has emerged today as the largest producer of milk in the world crossing 97 million tones annually. The organized dairy sector in India is largely dependent on buffalo milk as they contribute about 48 percent of the total milk produced in the country. Molecular genetic technologies will aid conventional methods of increasing production potential of indigenous buffaloes by augmenting genetic gain in a relatively shorter span of time. The candidate gene approach provides tools to search for causative polymorphisms affecting quantitative traits. Signal transducers and activators of transcription (STAT5A) and peroxisome peoliferator activated receptor gamma cofactor 1 alpha (PPARGC1A) are transcription factor encoding genes that are proved to be having candidate roles in milk production. The present work was carried out to study the polymorphism in STAT5A exon 7 and exon 10 and PPARGC1A partial exon 9 and intron 9 gene fragments by PCR-SSCP, PCR-RFLP and sequencing in Mehsana buffaloes.