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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 TRANSCRIPTOME PROFILING TO EVALUATE EFFECT OF HERBAL PLANT EXTRACT ON BULL SPERMATOZOA(Department of Animal Biotechnology College of Veterinary Science and Animal Husbandry Anand Agricultural University, Anand, 2019) Jignesh M. Italiya; Prakash G. KoringaThe mammalian ejaculated spermatozoa are highly differentiated terminal cells with an extremely compacted nucleus of haploid genome. The mammalian spermatozoon transfers coding as well as non-coding transcripts to the oocyte during fertilization. Spermatozoal transcript composition and expression levels are associated with spermatogenesis, functional parameters of spermatozoa, early embryonic development and pregnancy outcomes. Putranjiva roxburghii wall which is known as child’s amulet tree or child-life tree. Bryonia laciniosa is an herb, which has been included in Vrishya rasayana category in Ayurveda texts. It is also used traditionally as an aphrodisiac and pro-fertility compound. Hence this ethno-herb has immense potential of research in the field of infertility of either sex. On the basis of hypothesis that, any environmental disturbance in form of given treatment of herbal seed extract to spermatozoa that makes change in transcriptome profile, the current study is planned to establish the fact about effect of herbal preparations on sperm mortality, viability and RNA profiling.ThesisItem Open Access TOLL LIKE RECEPTORS (TLR) EXPRESSION IN MILK SOMATIC CELLS DURING MASTITIS IN CATTLE USING REAL-TIME PCR(AAU, Anand, 2011) GHADIGAONKAR, DINESH DILIP; Rank, D. N.The dairy sector in India has shown remarkable progress in the recent years and consequently the country has now become the largest milk producer and valueadded milk products in the world. Though the country is the largest producer of milk, milk production per animal is much less compared to agriculturally developed countries. One of the reasons for this less milk production per animal is loss of milk production capacity because of inflammation of the mammary gland or mastitis in dairy animals. Mastitis is defined as an inflammatory reaction of the parenchyma of the mammary glands to bacterial, chemical, thermal or mechanical injury which is characterized by a range of physical, chemical and usually bacteriological changes in the milk and pathological changes in the glandular tissue which is the most common and the most expensive disease of dairy cattle throughout the world. Mastitis is classified as subclinical and clinical mastitis. The cows that have subclinical mastitis are reservoirs of organisms that lead to infection to other cows. Most clinical cases start as subclinical; thus, controlling subclinical mastitis is the best way to reduce the clinical cases. Somatic cells consist of leukocytes and epithelial cells exfoliated from the mammary epithelium. Mastitis is associated with an influx of inflammatory cells; hence somatic cell count of milk increases. Enumeration of somatic cell counts and bacterial culture of milk has been suggested as a standard method for detecting subclinical udder infections in dairy cows. Genes have major impact on health status of animals. Genetic variability of mastitis resistance is well established in dairy cattle. Resistance to mastitis is a complex function involving various biological pathways, molecules and cells. Study of the expression of genes involved in mastitis resistance is major tool for early diagnosis of disease and genetic improvement to superior stock. Toll Like Receptors (TLR) are cell-surface receptors that recognizes a broad class of pathogen-associated molecular patterns, activates immune system, and induces the over expression of inflammatory factors, which participate in irmate immune responses to confer disease resistance. The bovine TLR genes have been studied in recent years. Hence the study was planned with the objective to investigate expression of TLR-2, TLR-4, TLR-9 in somatic cells in healthy and mastitic udder by Real Time PCR in cattle. The study was undertaken to assess different TLRs (TLR-2, TLR-4 and TLR- 9) in three cattle breeds namely Gir, Kankrej and Triple crossbred (Kankrej x Jersey x Holstein Friesian) with sub-clinical mastitis. A total of 65 lactating cows comprising 16 Gir, 29 Kankrej and 20 Triple crossbred animals were screened for presence of mastitis using Electronic Somatic Cell Counter and bacteriological culture examination. Total RNA was extracted from milk somatic cells from 15 positive and 6 healthy quarters from each breed using TRIZOL method. The RNA was treated with DNase enzyme to remove any traces of genomic DNA. cDNA was synthesized from RNA using Qiagen's Omniscript reverse transcriptase kit and random hexamer primers. The amplification of cDNA template of TLR 2, TLR 4 and TLR 9 genes was carried out using gene specific primers. Expression of TLR 2, TLR 4 and TLR 9 genes mRNA was quantified by Real Time PCR and analysed using Applied Biosystems 7500 SDS software. Relative expression study of these genes was carried out using GAPDH as internal control. Results indicated that there was upregulation of TLR 2, TLR4 and TLR9 gene expression in animals affected with subclinical mastitis compared to healthy animals. Targeted amplification of 421 bp TLR 2, 108 bp TLR 4 and 108 bp TLR 9 was confirmed by agarose gel electrophoresis. Prevalence of subclinical IMI was higher in Triple crosbred cows (65%) compared to Gir (50%) and Kankrej cows (27.59 %). The mean SCC of infected quarters was significantly higher than that of noninfected quarters (P < 0.05) in all three breeds. The average relative expression of all three genes i.e. TLR2, TLR4 and TLR9 was higher (ranged from 7 to 35 folds) in mammary gland with subclinical intramammary infection than those measured in the uninfected glands. The concomitant increase in somatic cell count and upregulation of TLR2, TLR4 and TLR9 gene expression was observed during subclinical mastitis in all three breeds. Comparison of SCC upregulation between breeds indicated that, there was no significant difference between breeds in the SCC in the diseased quarter during subclnical mastitis in Gir, Kankrej and triple crossbred cows. In Gir cows, TLR2 gene expressions level in diseased quarters was found to be upregulated with an average 10.54 (10.54 ± 7.12) fold compared to pooled healthy quarters. In Kankrej cows, TLR 2 gene expressions level in diseased quarters was found to be upregulated with an average 12.22 (12.22 ± 11.61) folds compared to pooled healthy quarters. In Triple crossbred cows, TLR 2 gene expression level in diseased quarters was found to be upregulated with an average 7.13 (7.13 ± 10.57) folds compared to pooled healthy quarters. In Gir cows, TLR 4 gene expressions level in all diseased quarters was found to be upregulated with an average 18.43 (18.43 ± 24.230) fold upregulation compared to pooled healthy quarters. In Kankrej animals, TLR 4 gene expressions level in diseased quarters was found to be upregulated with an average 31.59 (31.59 ± 18.74) folds compared to pooled healthy quarters. In Triple crossbred cows, TLR 4 gene expression level in diseased quarters was found to be upregulated with an average 23.817 (23.817 ± 27.6963) fold compared to pooled healthy quarters. In Gir cows, TLR 9 gene expression level in diseased quarters was found to be upregulated with an average 6.193 (6.193 ± 8.19) fold compared to healthy quarters. In Kankrej cows, TLR 9 gene expression level in diseased quarters was found to be upregulated with an average 5.44 (5.44 ± 8.14) folds compared to Pooled healthy quarters. In Triple crossbred cows, in all diseased quarters, TLR 9 gene expression level was found to be upregulated with an average 19.29 (19.29 ± 16.31) fold compared to pooled healthy quarters. During subclinical mastitis SCC was found to be positively correlated with the transcriptional activities of TLR2, TLR4 and TLR9 gene in Gir and Triple crossbred cow. In Kankrej cows TLR 2 and TLR 9 gene expressions were positively correlated with s e c but TLR 4 gene expression was not correlated with SCC. The level of infection as reflected by number of somatic cells had significant effect on level of upregulation in gene expression. However, there was no significant effect of a breed on level of upregulation of TLR 2, TLR 4 and TLR 9 gene expression.ThesisItem Open Access Evaluation of Gender Pre-selection through Sperm Enrichment Techniques Using Real-Time PCR in Bovines(AAU, Anand, 2013) ROY, LATISH CHANDRA; Panchal, M. T.The present work was undertaken to assess the efficiency of the classical X and Y sperm enrichment methods, viz., sperm swim-up, gravity sedimentation, Sephadex filtration, and discontinuous Percoll density gradient centrifiigation, using the modem molecular technique of Real-Time PCR or qPCR in buffalo and cow bulls, in the Department of Animal Biotechnology and Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science & AH, Anand Agricultural University, Anand. Total of 12 semen ejaculates six each from buffalo and cow bull, diluted at the rate of 1:10 with Tris-fructose-yolk- glycerol (TFYG), were collected from the breeding bulls stationed at Semen Station, Amul Research and Development Association (ARDA), Ode, Anand and subjected to the different enrichment techniques. In sperm swim-up four time bound fractions were collected using a special swim-up tube. Similarly with gravity sedimentation, three fractions, with Sephadex filtration four fractions and with discontinuous Percoll density gradient centrifugation the bottom sperm pellet was collected. DNA was extracted from all the collected fractions and the control semen samples and was further used for Real-Time PCR. Four different neat semen dilutions were taken as standards for the Real-Time PCR, viz., 100.00, 75.00, 50.00 and 25.00 per cent, consisting of 50.00, 37.50, 25.00 and 12.50 per cent X-chromosome bearing sperms,' respectively, considering the theoretical ratio of 1:1 for X and Y sperms in an ejaculate. With gravity sedimentation, the X-chromosome bearing sperm percentage in the three collected fractions of semen samples, ranged from 19.96 to 43.99 (31.90±4.44), 41.10 to 46.93 (45.01±1.32) and 38.90 to 48.07 (43.67±1.40) per cent, respectively and 21.09 to 51.32 (35.22±4.00), 35.46 to 51.32 (43.26±2.19) and 34.86 to 42.31 (39.19±1.14), respectively, in buffalo and cow bulls with an overall mean Xchromosome bearing sperm percentage of 40.19±2.08 and 39.22±1.67 in buffalo and cow bull semen, respectively. In the four semen fractions obtained post-filtration through Sephadex (G-lOO) gel, X-chromosome bearing sperm percentage values ranged from 42.51 to 52.08 (46.08±1.42), 42.75 to 52.51 (47.69±1.68), 41.43 to 50.20 (45.96±1.22) and 42.36 to 49.17 per cent (44.80±0.98) as compared to control semen samples having the range of 46.76 to 50.20 per cent (48.48±0.4) in buffalo bull and 27.82 to 48.00 (41.99±3.00), 33.02 to 52.59 (43.75±2.67), 35.09 to 52.79 (43.48±2.35) and 33.32 to 50.15 (42.50±2.20) per cent, compared to the control with the range of 45.94 to 50.11 (47.67±0.76) in the cow bull semen samples. The overall mean was obtained to be 46.13±0.67 and 42.93±1.21 per cent, in buffalo and cow bulls, respectively. The X-chromosome bearing sperm percentage in the three swim-up fractions retrieved ranged from 42.00 to 59.57 (48.62±2.60), 42.16 to 50.61 (45.91±1.29) and 30.13 to 51.93 (44.09±3.15) per cent, respectively, and 33.60 to 45.84 (42.30±1.80), 34.61 to 47.28 (42.86±1.84) and 31.23 to 62.58 (44.07±4.17) per cent, respectively, with an overall values of 46.21± 1.41 and 43.08±1.54, in buffalo and cow bulls, respectively. For control buffalo and cow bull semen samples the X-chromosome bearing sperm values ranged from 46.76 to 50.20 (48.48±0.48) and 45.94 to 50.11 (47.67±0.76) per cent, respectively. With discontinuous Percoll gradient centrifugation, the X-chromosome bearing sperm percentage obtained in the bottom pellet ranged from 43.79 to 51.83 (48.93±1.93) percent against the control value of 45.07 per cent in buffalo bulls and 48.20 to 56.89 (52.42±1.23) per cent, compared to the 50.56 per cent of the control, in cow bull semen samples. No detrimental effect was observed on individual motility of the sperms following any of the sperm enrichment procedures. None of the four methods evaluated proved efficient enough in altering the sex ratio of the sperms. No significant differences in X-chromosome bearing sperms were observed in any of the methods as compared to control, except in gravity sedimentation, where, a highly significant difference was found between the different fractions, both in buffalo and cow bulls.ThesisItem Open Access METHYLATION DETECTION IN H19 GENE IN BUFFALO [Bubalus bubalis) BY BISULFITE SEQUENCING(AAU, Anand, 2011) PATEL, HIREN MAVJIBHAI; Joshi, Chaitanya G.It is increasingly clear that translation of the genetic code into proteins is not the only way that our genes influence our growth, development and health. Another system of influence on the expression of genes is referred to as epigenetics. Any biochemical modification of the DNA or chromatin that can account for epigenetics gene expression system, only one candidate mechanism that is site-specific DNA methylation, has received experimental support to date. Methylation of DNA in mammalian cells occurs at cytosine residues in CpG dinucleotides. Improper or absence of DNA methylation of imprinted gene may lead to abnormal growth or cancerous condition. The best candidate for the epigenetic mark at the H19/Igf-2/Ins-2 locus is paternal-specific DNA methylation of the H19 and its 5' flank. Methylation status of H19 influences the expression of IGF-2. Keeping in consideration that methylation status ofH]9 influences the IGF2 expression, present study was carried out to detect methylation status in H19 promoter in Jaffarabadi, Surti and Mehsani buffalo by bisulfite sequencing. DNA extraction from blood was carried out by phenolxhloroform method from seven sample of each breed which were subjected to bisulfite treatment by sodium bisulfite. Quality and quantity of DNA was checked by ND-1000 spectrophotometer and 0.8% agarose gel electrophoresis. Locus specific primers for normal DNA and for bisulfite treated DNA were used to amplify a gene fragment of H19 in three breeds of Bubalus bubalis. Purification of PCR product was carried out by eluting desired PCR product from agarose gel by using by QIAquick® Gel Extraction Kit and later the PCR products were ligated in pTZ57R/T vector of InsT/Aclone TM kit (Fermentas). Ligated recombinant vector was transformed in competent E. coli (DH5-α) cells. Recombinant colonies were identified by blue-white screening and white conies were subjected to Ml3 colony PCR for confirmation of positive recombinant clones. White colony containing recombinant clone was grown in LB broth and recombinant plasmids were isolated using QIAprep® Spin Miniprep Kit and used for cycle sequencing, that were later processed for sequencing. Multiple sequence alignment was done for all 21 samples using BioEdit v 7.0.7.1, which revealed no difference between all samples. Four clones from each bisulfite treated samples were sequenced and subjected to methylation study by BiQ Analyzer and BISMA softwares. In H19, 20 CpGs were revealed in all three breeds and were subjected to methylation study. In all three breeds, highest methylation per cent was found to be 94.12, while in some sample it showed no methylation at any CpGs site. The overall methylation per cent of HI9 gene in Jaffarabadi, Surti and Mehsani buffaloes were observed to be 50.19, 70.85 and 52.24, respectively. The incidence of mean per cent methylation in H19 gene in all three breeds was found to be 57.36. Unexpected nucleotide conversion (T>C, A>G and G>A) and deletion (A and G) after bisulfite sequencing were observed. Methylation of non CpG cytosine was observed which were found at CpA and, only in one animal at CpT. In some case, three CpG out of twenty were not analyzed due to non specific conversion of CG>TA. For estimation of association of milk production traits with methylation pattern, animals were grouped into high and low group with respect to milk yield and milk fat percentage. Statistical analysis indicated that there was no significant difference in high and low group with respect to milk yield and milk fat per cent among methylation pattern in all three breeds. Screenings of more number of animals are needed to get reliable data for estimation of association of milk production traits with methylation pattern.ThesisItem Open Access METHYLATION DETECTION IN H19 GENE IN BUFFALO (BUBALUS BUBALIS) BY BISULFITE SEQUENCING(Anand Agricultural University, Anand, 2011) HIREN MAVJIBHAI PATEL; Dr. C. G. JoshiIt is increasingly clear that translation of the genetic code into proteins is not the only way that our genes influence our growth, development and health. Another system of influence on the expression of genes is referred to as epigenetics. Any biochemical modification of the DNA or chromatin that can account for epigenetics gene expression system, only one candidate mechanism that is site-specific DNA methylation, has received experimental support to date. Methylation of DNA in mammalian cells occurs at cytosine residues in CpG dinucleotides. Improper or absence of DNA methylation of imprinted gene may lead to abnormal growth or cancerous condition.ThesisItem Open Access “PROTEIN AND GENE POLYMORPHISM IN KAPPA CASEIN BY PCR-RFLP, SEQUENCING AND SDS-PAGE IN GOATS(Anand Agricultural University, Anand, 2011) SHETTY PRAKASH VITHAL; Dr. A.M. Pandeκ-casein (kappa-casein) is of special interest as a milk protein polymorphism due to its known relationship with milk quality and composition. A number of allelic variants have been identified in goats. In this study, genetic variants of the κ-casein gene were evaluated in Surti, Mehsani, Gohilwadi and Zalawadi goats breeds, using RFLP, DNA sequencing and SDS-PAGE.
