Loading...
Kerala Veterinary and Animal Sciences University, Wayanad
Browse
5 results
Search Results
ThesisItem Open Access EXPRESSION PROFILING OF MICRORNA IN LIPOPOLYSACCHARIDE CHALLENGED PERIPHERAL BLOOD MONONUCLEAR CELLS OF CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) AKSHARA B. VENU; Dr. Divya P.DMicroRNAs are endogenous, small non-coding RNAs approximately 20-22 nucleotides long which play a key role in gene regulation by affecting translation and transcription mechanisms. The present study was undertaken to analyse the differences in the microRNA (miRNA) expression in (LPS) challenged peripheral blood mononuclear cells (PBMCs) of cattle. The current research work was carried out using the isolated blood samples from apparently healthy female crossbred cattle maintained at the University Livestock Farm and Fodder Research and Development Scheme, Mannuthy. Isolation of PBMCs were done by density gradient centrifugation using HisepTM lymphocyte separation medium 1077.Expression of miRNAs viz; bta-miR-451, bta-miR-107, bta-miR-93 and bta-miR-125a, whose expression was found to be varying in the LPS treated and untreated bovine PBMCs by miRNAome analysis of previous studies was validated in the present study by qRT-PCR assay. Insilico analysis was done using various online tools for the prediction of target genes of real time PCR validated miRNAs, gene ontology analysis and also to study the cellular pathways associated to the target genes. Targets of each miRNAs were predicted using the online target prediction programme TargetScan Human 8.0. Through the use of the online DAVID bioinformatics tool, gene ontology analysis of the real time PCR validated miRNAs was carried out. Predicted targets for each miRNAs were annotated to biological process, molecular function and cellular component categories. The pathway analysis programme (KEGG) of the DAVID database was utilized to analyze and interpret the pathways associated with the predicted targets of real time PCR verified miRNAs. The study also included the assessment of cytokines associated to the cellular pathways influenced by the target genes of real time PCR validated miRNAs in the supernatants of PBMC cultures from both treatment and control groups by ELISA. The expression of miRNAs; bta-miR-451and bta-miR-107 was found to up regulated while bta-miR-93 and bta-miR-125a showed decrease in their expression in LPS treated PBMCs when compared to control cells. The results of real time PCR validation of aforementioned miRNAs expression were in accordance with the findings of miRNAome analysis. Besides, significant enrichment of target genes of the real time validated miRNAs was noticed in many immune related GO terms as well as in critical immune associated cellular pathways. Measurement of cytokine, TNF α revealed a significantly higher level in LPS treated PBMCs when compared to LPS untreated cells. The findings of the current study will help in understanding the role of miRNAs in LPS mediated immune responses in cattle.ThesisItem Open Access CHARACTERISATION OF ACID SOLUBLE COLLAGEN FROM DAGGERTOOTH PIKE CONGER (MURAENESOX CINEREUS) AND ITS APPLICATION IN WOUND HEALING(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) NIMNA AJAY; Dr. Varuna P. PanickerCollagen, the abundant structural protein in the extracellular matrix has wide application in medical, tissue engineering and pharmaceutical industry. The present study was conducted to characterize and evaluate the wound healing potential of isolated ASC from Daggertooth pike conger (Muraenesox cinereus).Conger eels were collected from Kochi harbour and pre-treatment for removal of fats and non-collagenous proteins. ASC was isolated by treating with 0.5 M acetic acid for 3 days and supernatants were collected. Protein precipitation was carried out by adding NaCl to final concentration of 2.3 M. Precipitated samples were dissolved in 0.5 M acetic acid and dialysed against 0.1 M acetic acid for one day and in distilled water until neutral pH was obtained. Yield of ASC was 12.78 and 31.95 per cent on wet and drymatter basis respectively. ASC on Lowry’s test showed a concentration of 8.8µg/ml. On electrophoretic separation revealed the presence of characteristic α (2 α1 and α2), β, γ subunits similar to that of type I calf collagen. An absorption peak of 221 nm characteristic to triple helix was observed on UV-visible spectroscopy. FTIR spectrum of ASC showed Amide bands similar to that of collagen. Analysis for secondary structure, by CD spectroscopy revealed a CD spectra with positive absorption maximum at 222 nm, and negative at 214 nm. Denaturation temperature of the extract was obtained at 38.2℃ which in turn confirm that collagen can withstand very high temperature. Solubility assay revealed a maximum solubility at low salt concentration and acidic pH. Hydroxyproline concentration in the extract was 9.9g. Invivo wound healing study showed that group treated with paste has increased wound healing compared to gel and control group. Results were confirmed by histopathology, immunohistochemistry and relative quantification of selected growth factors and cytokines. On histopathological evaluation increased fibroblast proliferation, collagen deposit and re-epithelisation were observed on collagen treated group. Difference in the collagen deposition in response to treatment was studied using staining like Picrosirus and Masson’s trichome. Angiogenesis was studied by immunohistochemistry using CD31 antibody. Observations on histopathology and immunohistochemistry could correlate with the gene expression study of TGFβ-1, VEGF, IL-10 and TNF-α. Upregulation of TGFβ-1, VEGF and IL-10 and downregulation of TNF-α was observed in collagen treated groups which might be due to the wound healing potential of collagen.ThesisItem Open Access MOLECULAR STUDIES ON HEAT SHOCK PROTEIN 70 GENE IN DOMESTIC FOWL AND DUCK(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) LIJO JOHN; Sisilamma GeorgeA study has been carried out to analyse the promoter and 3’UTR of hsp70gene in different breeds of chicken (Naked neck (NaNa), Kadaknath (KAD), White Leghorn (WL), Rhode Island Red (RIR) and White Plymouth Rock (WPR)) and duck (Kuttanadu (KUT) and White Pekin (WP)). Plasma corticosterone level was also estimated in two different (summer and rainy) seasons. Promoter and 3’ UTR of Hsp70 gene were amplified by PCR and the products were sequenced. Sequence analysis of the promoter region showed 100% homology between three breeds of chicken, WL, RIR and KAD and the duck breeds. These breeds exhibited only 99.7% identity with WPR and NaNa Sequence alignment (with Gene bank accession no. J02579) revealed a point mutation in the CAAT box, where an ‘A’ is deleted at position -312 in all the breeds of chicken and duck except NaNa. Another ‘A’ deletion could also be detected at position -329 from a heat shock element in WPR. In all the breeds under study, TATA box (TATAA) was found at position -134. Three inverted CAAT boxes, ‘ATTG’ and two variants of GC boxes, one having GGCG motif (6 numbers) and another having GGGCGG motif (2 numbers) were identified in both chicken and duck sequences. Two variants of consensus heat shock elements (HSE), NGAAN (single unit) and ‘NGAAGAAN’ (double unit) were detected in the promoter region of all breeds of chicken and duck under study except WPR, in which a point mutation (‘A’ deletion) was noted in one of the double units. Restriction analysis showed that only Bgl І has a site in the amplified region of the promoter of both the species. Due to the presences of point mutations, three alleles for the promoter region of the gene could be detected. Sequence analysis of 3’ UTR showed varying levels of sequence polymorphism between chicken breeds, where only 80 to 95.8 % identity could be observed. Between duck breeds, KUT and WP, 97.7 % identity was observed. Analysis between chicken and duck revealed 79 to 99.5% identity where 97 to 99.5% identity was observed between duck breeds and WPR. Two consensus, CAAC sequence and a variant of poly adenylation signal sequence could be identified in 3’ UTR. A variant of poly adenylation signal sequence, TATAAA could be identified in all breeds except RIR, in which two point mutations (transversion) were observed (TAAAAA) when compared to the consensus sequence (AATAAA). A second poly adenylation signal sequence, which was again a variant (AATAAT) was detected only in NaNa. The number and position of CAAC motifs varied (2 to 4 numbers) between species and breeds under study. In both the species, rather than a consensus sequence, a variant of K box (GGTGAT) and Brd box (TGCTTA) could be identified. Several AT (AU in mRNA) rich regions could be identified in the 3’ UTR of both chicken ad duck breeds. However, an additional ATATA motif is also detected in RIR and NaNa. Restriction enzyme analysis of 3’ UTR revealed that NaNa, RIR and WL have no cutting site for any of the common enzymes while a single cutting site for Bgl ІІ was observed in KAD, showing two alleles in chicken breeds. Duck breeds also showed two alleles where the enzyme, Bam HI has a restriction site at different positions. Plasma corticosterone level in chicken and duck showed considerable variation in different breeds of chicken both in summer and rainy seasons. Among the chicken breeds WPR showed the lowest level of corticosterone in summer season, which did not differ significantly from that of rainy season. Comparison between seasons showed a highly significant (P<0.01) increase in summer in all breeds except WPR. Duck breeds also showed a similar trend. Significant (P<0.01) increase in the plasma corticosterone level was noticed in summer. However, between breeds, no significant variation was observed in each season. Hormone responsive elements (HRE) for corticosterone could not be detected in the promoter region of any of the breeds under study. Any correlation between the sequences and the plasma corticosterone level could not be detected in both chicken and duck breeds under study. Although, two point mutations were detected in the promoter region of WPR, it could not be correlated with the plasma corticosterone level.ThesisItem Open Access METABOLIC PROFILE OF CROSSBRED DAIRY COWS DURING TRANSITION PERIOD(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2017) ANISHA, J. PERUMBILLY; Shynu, M.Dairy cows are considered to be most prone to diseases during the period from late pregnancy to onset of lactation, i.e. during the transition period. The present investigation was carried out in blood/serum collected at fortnightly intervals from 15 crossbred dairy cows, from eight weeks before the predicted date of calving till eight weeks after calving, with the objectives of generating a metabolic profile and for evaluating the antioxidant status. Concentrations of glucose, NEFA, BHB, cholesterol, urea, albumin, ceruloplasmin and haptoglobin were determined; differential leukocyte count was done; and assessment of oxidative stress was also performed. The mean concentration of glucose (47.35±1.32 mg/dL) and cholesterol (95.83± 3.62 mg/dL) during transition period was significantly lower than pre and post-transition period. NEFA (0.576 ± 0.08 mmol/L) and BHB (0.638 ± 0.05 mmol/L) concentrations reported significant increase during transition when compared to pre-transition period. Concentration of indicators of protein status viz. albumin and urea were 3.39 ± 0.09 g/dL and 12.26 ± 0.66 mg/dL respectively during transition period and did not differ significantly from pre or post-transition period. Out of the two acute phase proteins measured, ceruloplasmin did not show significant variation during the study period, but a significant increase was shown by haptoglobin during transition period. The level of MDA, an indicator of oxidative stress was higher during transition period, indicating significant oxidative stress during the period. TAS did not show significant change but the antioxidant status of the animals could not be considered optimum, as eleven out of fifteen animals exhibited diseases albeit transient during the period. A significant increase in the number of neutrophils and monocytes and a highly significant decrease in the number of lymphocytes observed during transition period could be due to the influence of corticosteroids. A comprehensive study involving more number of transition animals, maintained under different managemental conditions shall help in establishing reference intervals for various analytes during period.ThesisItem Open Access ISOLATION AND CHARACTERIZATION OF LACTOFERRIN FROM COLOSTRUM OF GOATS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2017) Sinchu Vijayan; UMA, R.Lactoferrin, an 80 k Da iron-binding protein, primarily present in milk, is well known for its multifunctional properties such as antibacterial, antifungal, antiviral, antioxidant, immunomodulatory and anticancer activities. The present study focussed on the isolation and characterization of lactoferrin from the colostrum of Malabari, Attappady Black and crossbred goats of Kerala as well as assessment of the antimicrobial potential of the lactoferrin isolated. Colostrum samples collected from the three goat breeds maintained at University Goat and Sheep Farm, College of Veterinary and Animal Sciences, Mannuthy were processed to remove fat globules and casein. The whey obtained after processing was fractionated with ammonium sulphate to remove globulins from the sample. Fraction containing albumin and remaining proteins including lactoferrin was separated out, dialysed against equilibration buffer, loaded on to CM- Sephadex C-50 cation exchanger column and eluted with a step gradient of 0.4, 0.6 and 0.8 M NaCl. The fractions with high OD280 values were analysed using 12 per cent SDS-PAGE to identify their protein components in comparison with standard protein. The protein fractions with high absorbance at 280nm, eluted with 0.6M NaCl, could be visualised as a single 80 kDa Coomassie Brilliant Blue-stained band. The total iron content in the isolated lactoferrin samples was estimated by atomic absorption spectrophotometry and it was found to be 820 ppm for Malabari and Attappady Black lactoferrin whereas 1100 ppm for crossbred goat lactoferrin. The concentration of Malabari lactoferrin (mgLf), Attappady Black lactoferrin (agLf) and crossbred goat (cgLf) as estimated by Lowry’s method was found to be 10.94, 12.93 and 11.22mg /L of colostrum respectively. These isolated samples of caprine lactoferrin were found effective to inhibit the growth of both Gram-positive and negative organisms. The assay depicted that the minimum inhibitory concentration (MIC) of mgLf and agLf against E. coli and S. aureus was 275µg/mL and 550µg/mL respectively. The MIC of cgLf against both E. coli and S. aureus was found to be 550µg/mL. The indigenous as well as crossbred goat lactoferrin exhibited the same intensity of antibacterial activity against Gram-positive bacteria. Against Gram-negative organism, lactoferrin of indigenous goats were found to be more potent when compared to the crossbred goat lactoferrin.
