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Theses (M.Sc.)
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ThesisItem Open Access DEVELOPMENT OF A QUANTITATIVE COLORIMETRIC STRIP FOR DETERMINING THE PLASMA GLUCOSE AND FREE FATTY ACID LEVELS IN INDIGENOUS COW BREEDS AND MURRAH BUFFALO(ICAR-NDRI, KARNAL, 2022) V MOUNICA; Suneel Kumar OnteruGlucose and free fatty acids (FFA) are the basic energy sources in ruminants. It is well known that high plasma free fatty acids shows the possibility of negative energy balance. The levels of these metabolites indicate the nutritional status of ruminants. However, the plasma glucose and FFA levels of indigenous cattle and buffaloes at different stages of growth are not available. Hence, the present project was targeted to establish the plasma glucose and FFA levels in calves, prepubertal, and pubertal heifers of Gir, Sahiwal, Tharparkar, Karan Fries, and Murrah buffalo, and also, to develop a quantitative colorimetric strip to detect plasma glucose and FFA. The plasma glucose levels were estimated using three different methods, including glucometer, O-toluidine and a new paper based test strip method, which is dependent on the principle of the in-situ synthesis of gold nanoparticles by the reducing ability of glucose. Among them, there was a higher correlation (0.65) between glucometer and a paper based strip method. On the basis of glucometer method, the glucose levels ranged from 39-118 mg/dl, 30-124 mg/dl, 23-113 mg/dl, 46- 94mg/dl, 54-182 mg/dl in Tharparkar, Gir, Sahiwal, KF cattle breeds and Murrah buffalo, respectively. Based on a paper based strip method, the glucose levels ranged from 58-105 mg/dl, 38-67 mg/dl, 32-68 mg/dl, 30-65 mg/dl, 38-65 mg/dl in Tharparkar, Gir, Sahiwal, KF and Murrah buffalo, respectively. Considering a reported range (37-71 mg/dl) of glucose levels in cattle in general, paper based strip method appears to be more applicable for cows and buffaloes than commercially available glucometer method. There was a significantly lower plasma glucose level (estimated by glucometer) in pubertal heifers than pre-pubertal and female calves in KF breed. Comparative analysis of plasma glucose levels among different breeds found that Tharparkar has higher plasma glucose levels than the other selected breeds. The plasma FFA levels were estimated using Gas chromatography, and only short chain fatty acids (acetic acid, propionic acid and butyric acid) were identified in the plasma samples of calves, pre-pubertal and pubertal heifers of Tharparkar, Gir, Sahiwal and KF cattle and Murrah buffalo. The propionic acid concentration appears to be higher in plasma samples, except in the pubertal heifers of Tharparkar and Murrah, in which butyric acid concentration appears to be higher. Comparative analysis of plasma propionic acid levels among different cattle breeds and Murrah buffalo showed that female calves and prepubertal heifers of Sahiwal had higher propionic acid levels than other cattle breeds and Murrah buffalo. In addition, a paper based strip was developed to quantify plasma glucose and FFA. However, further research is needed to enhance the strip sensitivity for easy detection.ThesisItem Open Access EVALUATION OF B-COMPLEX VITAMINS BIOSYNTHESIS IN LIMOSILACTOBACILLUS FERMENTUM (MTCC 5898)(ICAR-NDRI, KARNAL, 2022) KARADAGATLA SUMAN; RAJEEV KAPILAVitamins are typically categorized as fat‐soluble vitamins, and water-soluble vitamins which include series of B-vitamins thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), biotin (B7), folic acid (B9), and cobalamin (B12). These vitamins serve as coenzymes that typically transport specific chemical groups to many biochemical reactions. (Baku et al., 2001) has reviewed the biochemical pathways involved in B‐vitamin biosynthesis by probiotic bacteria. Bacterially synthesized B-vitamins could supplant the expensive chemical production of these vitamins to enrich food or be improved for in situ fortification of fermented foods. Much research has been conducted in recent years to elucidate the biosynthetic pathways of these vitamins in a number of microorganisms. As Limosiactobacillus fermentum (MTCC 5898) is safe and nontoxic and may be considered for functional food application after clinical testing. The therapeutic management of various diseases associated with B-complex vitamins without any side effects remains a challenge. In response, there is a growing interest in evaluating B-complex vitamins biosynthesis in probiotic bacteria. which are seen to be human friendly and to have negligible side effects. By considering these facts, present study was undertaken to evaluate B-complex vitamins Biosynthesis genes in Limosilactobacillus fermentum on the progression of Bcomplex vitamins biosynthesis, as whole genome sequence of Lm. fermentum is available it was annotated using RAST rapid annotation subsystem technology tool, finding out genes associated with vitamins biosynthesis in genome, and other complete Lm. fermentum strains were downloaded from NCBI data base and all sequences were annotated for finding out the same as stated above , then from all sequences data were collected and multiple sequence alignment were performed and after this analysis found out that B-complex vitamins like 1.Thiamine2.Folate,3.pyridoxin .were considered for expression after target enzymes which are involved in the pathways were found out by genome scale metabolic modelling along with enzyme structures were predicted and compared with other strains structure, then primers were designed for target genes by using Primer 3 plus software , then bacterial culture were activated ,RNA were extracted BY trizol method , c DNA synthesized by RevertAid kit ,after analysing primer expression by running gel electrophoresis ,performing quantitive Real time PCR CT values are collected and results are compared against housekeeping gene as control and graphs were plotted by using prism graph pad by performing one way a nova . after observing all the parameters that B-complex vitamins 1. Thiamine , 2. Pyridoxin, 3. Folate, were synthesized by the potential probiotic strain Limosilactobacillus fermentum MTCC 5898.ThesisItem Open Access EVALUATION OF ANTIDIABETIC POTENTIAL OF MORINGA OLEIFERA ENRICHED PROCESSED CHEESE SPREAD(ICAR-NDRI, KARNAL, 2022) DESHAM AKSHITHA; SUMAN KAPILADiabetes is an endocrine and metabolic disease occurs due to defect either in insulin secretion or in its action or both. Long term complication of diabetes is also associated with several risk factors such as chronic kidney disease, cardiovascular complication, hyperlipidaemia, and obesity. To prevent or control high blood glucose levels of an individual, different approaches are being used on the basis of lifestyle of people i.e., diet and exercise and pharmacological interventions. Long term treatment of diabetes and its complications can cause enormous cost for society and currently drugs used for treatment are expensive with some undesirable side-effects. The therapeutic management of diabetes without any side effects remains a challenge. In response, there is a growing interest in evaluating herbal remedies, which are seen to be less toxic and to have negligible side effects. By considering these facts, present study was undertaken to evaluate suppressive effect of Moringa oleifera enrinched cheese spread on the progression of diabetes and its effects in streptozotocin (STZ) and Nicotinamide induced diabetes in rats. Feeding of Moringa cheese spread suppressed the elevation of blood glucose, glycosylated haemoglobin (HbA1C), insulin, lipids i.e. total cholesterol, triglycerides, low density lipoprotein (LDL)-cholesterol, very low density lipoprotein (VLDL)-cholesterol. It also showed lower levels of oxidative stress markers such as thiobarbituric acid reactive species (TBARS) and higher levels of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase in RBCs. Kidney function tests i.e., plasma urea, creatinine and uric acid were lower in Moringa fortified cheese spread fed animals. Liver function tests i.e., aspartate aminotransferase (AST), alanine aminotransferase (ALT) were lower in MFC fed animals. It has also been observed that there was significant decrease in glucose-6-phosphatase (G- 6-Pase), GLUT-2 and cyp8b1 gene expression levels in MFC fed animals. Histopathological pancreatic sections of diabetic control and diabetic fed with normal cheese shows rupture of islets cells whereas diabetic group fed with MFC has shown regeneration of islets cells. From the above results we conclude that Moringa Oleifera enriched cheese spread has anti-oxidative properties responsible for regeneration of islets cells of pancreas, enhancing the release of insulin leading to decrease in high blood glucose levels in diabetic condition which further relieve from other biological complexities in body. This Moringa Oleifera enriched cheese spread having good palatability can be consumed by diabetic patients.ThesisItem Open Access ASSESSMENT OF ENCAPSULATED ĸ-CASEIN HYDROLYSATES FOR ITS DPP-IV INHIBITORY ACTIVITY(ICAR-NDRI, KARNAL, 2022) NITIN MAHAL; SUNITA MEENADipeptidyl Peptidase-IV (DPP-IV) is a dimeric, transmembrane glycoprotein and exopeptidase that is expressed on the surface of lymphocytes and monocytes, as well as in tissues from the pancreas, kidney, liver, and gastrointestinal tract. It is involved in the cleavage of a variety of substrates including the incretin hormones (viz. Glucagon like peptide-1 and Glucose-dependent insulinotropic polypeptide). GLP-1 binds to its respective GLP-1 receptors of pancreatic β-cells and leads to β-cell proliferation, increase in insulin gene expression and insulin secretion. In diabetes, increase in glucose level leads to the glucotoxicity which destroys pancreatic beta cells, and leads to decreased insulin level and further increasing blood glucose level. Inhibition of DPP-IV activity is one of the strategies for the treatment of type-2 diabetes. In recent years, peptides derived from a variety of dietary proteins have been reported to exhibit DPP-IV inhibitory activity. To keep their bioactivity intact, these peptides should also be protected from the gastrointestinal damage. Therefore, the present study was conducted to compare the antidiabetic potential of the Emulsified encapsulated hydrolysates (EEH) and Spray-dried encapsulated hydrolysates (SDEH) with non-encapsulated hydrolysates (NEH). IC50 value of non-encapsulated hydrolysates came out to be 84.70μg/ml. Simulated gastrointestinal digestion was done for NEH, EEH and SDEH. The digested samples were assessed for their bioaccessibility by transwell assay. RP-HPLC shows many similar peptides peaks for the samples collected from both apical and basal chamber of transwell suggesting transportation of NEH, EEH and SDEH hydrolysates through HT-29 cells. Peptide peaks showing similar retention time for NEH sample collected from both apical and basal chambers were 1.753, 2.130, 4.464, 4.562, 4.776, 7.834, and 7.864. Similarly, for EEH, identical peptide peaks from apical and basal chambers were 2.2, 4.193, 4.38, 4.836, 7.974, 7.223, and for SDEH identical peptide peaks were 4.238, 4.577, 5.534, 5.774, 6.184, 6.827, 8.710. Hence, NEH, EEH and SDEH are bioaccessible. Sitagliptin, NEH, EEH, SDEH and Milk are administered orally at a dose of 300 mg/kg body weight and their impact was also evaluated in Nicotinamide-Streptozotocin induced type-2 diabetic rats in a study of 4 weeks. Diabetic control (D-Control) showed a significant (p<0.05) decrease in the body weight, HDL-C, insulin, GLP-1 levels, SOD and catalase activity and there was a significant (p<0.05) increase in the blood glucose, glycated Hb, TGs, TC, VLDL-C, LDL-C, atherogenic index and liver enzymes (ALT and AST). Oral doses of the hydrolysates helped in improvement in the diabetic condition in experimental rats. There was a significant (p<0.05) decrease in the blood glucose and glycated Hb level in diabetic rats fed with NEH, EEH and SDEH as compared to diabetic control. EEH feeding normalise the blood glucose and glycated Hb levels. The level of plasma insulin was increased in the case of diabetic rats fed with hydrolysates and the level of GLP-1 was significantly increased by EEH feeding. There was significant decrease in the plasma DPP-IV enzyme in the groups treated with Sitagliptin, NEH, EEH, SDEH and Milk as compared to diabetic control group. Feeding of NEH, EEH, SDEH and Milk in diabetic rats displayed a significant (p<0.05) decrease in TGs, VLDL-C, LDL-C and atherogenic index and increase in HDL-C as compared to diabetic control. EEH was able to normalize lipid profile parameters except HDL-C. The level of liver enzymes viz., ALT and AST were significantly (p<0.05) decreased by NEH, EEH and SDEH administration. Feeding of hydrolysates significantly improved the SOD and catalase activity as compared to diabetic control group. Thus, it is concluded that, NEH, EEH and SDEH and milk exhibited anti-diabetic potential due to its DPP-IV inhibitory activity. Among all groups, hydrolysates were better and among hydrolysates encapsulated hydrolysates (EEH and SDEH) have displayed comparatively better anti-diabetic potential, which can be credited to its encapsulation.ThesisItem Open Access IN SILICO IDENTIFICATION AND CHARACTERIZATION OF EPITOPE-BASED PEPTIDES OF BUFFALO HSP70 AND TLR4(ICAR-NDRI, KARNAL, 2022) SHIPRA CHAUHAN; VARIJ NAYANIndia holds the top spot with over 109.85 million buffaloes worldwide, although estrus identification remains a significant bottleneck due to the less obvious estrus indicators in buffaloes. There are many ways to identify estrus, but they are typically time-consuming, difficult, and ineffective. In light of this, the development of future immunodiagnostic tools and the positive prospective future of structure-based computational biology in the field of animal sciences, two proteins, HSP70 and TLR4, are expressed more strongly during the estrus phase of the buffalo estrous cycle than during the other stages, according to past scientific reports. To identify estrus, HSP70 and TLR4 can be used as potential biomarkers. Consequently, for a sustainable system of animal production and to prevent conflict between animals and humans in terms of disease transmission and other potential methods. To understand the various concepts and dynamics of immunology and systems biology, new and novel technologies linking immunology and "-omics" techniques are therefore highly required. For example, in silico epitope-based peptide synthesis is an effective method for designing future immunodiagnostic tools, vaccines, and estrus identification for maintaining the overall health of buffalo and its reproductive efficiency. Therefore, the goal of the current study is to transform -omics data into diagnostics that can then be used to create anti-peptide antibodies for the development of future immunoreagents and immunodiagnostic tools for diagnosing diseases. Additionally, the identification of estrus and the study of biological interactions between proteins and their ligands offer a tremendously promising future in the fields of in silico pharmacology and drug designing for veterinary medicine and animal health welfare.ThesisItem Open Access EVALUATION OF CASEIN DERIVED BIO-DEGRADABLE FILMS FOR WOUND DRESSING(ICAR-NDRI, KARNAL, 2022) SHAIK ROSHAN MOHIDDIN; RAJEEV KAPILACasein-based antibacterial films are attractive materials for wound dressing application because they possess chemical, mechanical, exudate absorption, drug delivery, antibacterial, and biocompatible properties required to support wound healing. Generally, gauze, lint and cotton wool are often used as wound dressings. However, the major drawback of such materials is painful removal which can cause damage to healed tissue. Their opaqueness also becomes a crucial issue for sensitive wound applications that requires visualization-based diagnosis and treatment methods. So, proteins like casein can be formulated into films, hydrogels and electro spun fibers for topical antibacterial therapy. The aim of this study was to to compare the properties of casein derived biodegradable films obtained from cow, buffalo and goat along with the in vivo wound healing trials on rats. For this study we had isolated casein from cow, buffalo, goat milk and prepared casein films using different cross-linking agents. The different cross-linkers used were aldehyde (glutaraldehyde), polysaccharide (alginate dialdehyde) and enzyme (tyrosinase). Films were made with and without glycerol. The bio-film properties were studied by comparing all the films made among the species along with different cross-linkers. Preparation of milk casein films using cross-linking agents (glutaraldehyde, alginate dialdehyde and tyrosinase) significantly (p<0.01) increased the water absorption, film expansion and tensile strength, respectively. Use of alginate dialdehyde in preparation of goat casein films significantly (p<0.01) improved the water absorption, oxygen permeability and moisture permeation properties. There is no major difference in film expansion ratio of casein films prepared from cow and goat milk using glycerol irrespective of the cross-linking agent used. Though this feature was significantly higher as compared to buffalo films. Biofilms prepared with buffalo casein have least water absorption and high moisture permeation in comparison to cow and goat films. Drug release property had revealed that there is slow and sustainable release of drug observed in case of alginate and tyrosinase cross-linked casein films. The prepared films were smooth, flexible, and transparent, which can aid in inspecting wounds without removing the dressing. The Goat casein cross linked with alginate dialdehyde had shown better properties and selected for in vivo trials. Herein, we fabricated films composed of casein and alginate dialdehyde (AD) loaded with gentamicin sulfate (GS) for application as a wound healing aid. In disk diffusion assay, the alginate cross-linked goat casein films demonstrated excellent antibacterial effect against E. coli, S. aureus. Overall, the findings suggest that GS-loaded goat casein alginate dialdehyde (CAD) films hold potential for further development as antibacterial wound dressing material.ThesisItem Open Access CHARACTERIZATION OF MUCINS ASSOCIATED WITH THE TYPICAL FERN PATTERNS OF BUFFALO SALIVA AT ESTRUS(ICAR-NDRI, KARNAL, 2022) MEET THUMAR; DHEER SINGHEstrus detection is a major problem in buffalo farming due to various physiological reasons and also because of the lower efficiency of the conventional estrus detection method, such as observing estrus-specific behavioural signs in farm conditions. As a result, detecting an estrus event is missed several times in buffaloes, especially during the summer season. Therefore, it is necessary to develop a simple and effective new estrus detection technique for buffaloes. Observing the typical fern-like crystallization patterns of buffalo saliva is one such estrus identification technique. Although fern-like crystallization patterns of the dried saliva are associated with the estrus stage, it is necessary to identify the molecules responsible for causing the typical fern-like patterns of buffalo saliva at the estrus stage for the development of a colour reaction. Hence, the present research work was focused on the identification of the molecules associated with the formation of the typical fern pattern, as it might help in developing a colourbased estrus detection kit. For this, the dried buffalo saliva smears containing typical and atypical fern pattern areas were scratched and the scratched powders were subjected to SDS – PAGE, followed by either silver staining or Coomassie brilliant blue staining. Two band shifts were observed in the SDS - PAGE, one was at 130 kDa molecular weight and the other was observed at 55-70 kDa molecular weight. The protein associated with the formation of a typical fern pattern was found to have a higher molecular weight as compared to the same protein associated with the formation of an atypical fern pattern. To identify the proteins expected to have Post – Translational Modifications (PTMs), MALDI MS/MS analysis was done from the SDS-PAGE gel plugs for two protein bands representing typical fern-like patterns and two protein bands representing atypical fern patterns. Analysis of four protein bands by MALDI MS/MS identified that Mucin – 1 (MW: 58 kDa) was the common protein found in the solutions of scratched powder of typical and atypical fern patterns. Considering a band shift at the 58 kDa region on SDS-PAGE, some, PTMs perhaps carbohydrate binding was expected. To confirm this expectation, SDS – PAGE gel was stained with ‘stains all’ dye and it was found that the protein associated with the formation of the typical fern pattern was glycosylated in nature as compared to the same protein which was associated with the formation of atypical fern pattern. To confirm this, three probable carbohydrates (not shown in the thesis because of the planning of a patent or technology to release) were estimated in the solution containing the scratch powder of the typical and the solution containing the scratch powder of atypical fern patterns obtained from the glass slide, on which buffalo saliva was smeared and dried. Also, the same carbohydrates were estimated in the cell-free saliva of the animals at the estrus stage and diestrus stage. The scratch solution of the typical fern pattern had a higher concentration of selected carbohydrates as compared to that of the scratch solution of the atypical fern pattern. Also, the concentrations of the same carbohydrates were higher in the saliva of the animals at the estrus stage than in the saliva of the animals in the diestrus stage. The conclusion of the research work is, Mucin 1 protein is one of the several proteins associated with the formation of the typical and atypical salivary fern pattern. But the Mucin 1 associated with the formation of the typical salivary fern pattern were glycosylated and those associated with the formation of the atypical salivary fern pattern were not. The difference in the concentration of these carbohydrates can be exploited further to develop a colour-based estrus detection kit.ThesisItem Open Access Panchgavya mediated zinc oxide nanoparticles: biogenic synthesis, characterization and in vitro antimicrobial activity against bovine mastitis pathogens(ICAR-SRS-NDRI, KARNAL, 2022) Sentinaro; MAMTA CHAUHANThis study aimed to investigate the synthesis, characterization and evaluation of antibacterial activity of ZnO nanoparticles (ZnO NPs) against bovine mastitis. Panchgavya was used as a biological reducing and capping agent for the synthesis of ZnO NPs from Zinc acetate dihydrate. Characterization of ZnO NPs by UV- Vis analysis showed absorption band at 320 nm thereby confirming ZnO NPs formation. The morphology of NPs as shown by Scanning Electron Microscopy was spherical with average size of 43 nm. The average size of ZnO NPs using Dynamic Light Scattering was 204.6 nm with zeta potential of -43.8 mV. The X-ray Diffraction revealed the crystalline structure of biogenic ZnO NPs having hexagonal pattern with crystallite size of 12 nm. The Fourier Transform Infrared Spectroscopy demonstrated the presence of phenol, carboxylic acids and aromatic amines on ZnO NPs surface that act as capping and stabilizing agent. The antimicrobial potential of ZnO NPs was examined using well diffusion method against two bacterial isolates from bovine mastitis milk which were identified through 16S rRNA gene sequencing as Staphylococcus aureus and Enterococcus faecalis. Panchgavya mediated biogenic ZnO NPs demonstrated antibacterial properties against Staphylococcus aureus and Enterococcus faecalis bacteria isolated from bovine mastitis as observed by well diffusion assay, Minimal Inhibition Concentration (MIC) and Minimum Bactericidal Concentration (MBC) tests. The zone of inhibition for 100 μg and 50 μg of ZnO NPs against S. aureus was 14±0.57 and 8±0.5 mm respectively. The zone of inhibition for 1000, 800 and 500 μg of ZnO NPs against E. faecalis was 14±0.57, 13±0.5 and 12±0.5 mm respectively. The MIC and MBC of ZnO NPs against S. aureus and E. faecalis were 250 and 500 μg/ml respectively. These in vitro results indicate that the biogenic ZnO NPs present antimicrobial activity against S. aureus and E. faecalis of bovine mastitis origin.ThesisItem Open Access Biogenic synthesis and characterization of panchgavya mediated silver nanoparticles for evaluation of antimicrobial activity in vitro against neonatal calf diarrhea(ICAR-SRS-NDRI, KARNAL, 2022) Pde, Lasuki; MAMTA CHAUHANThis study was performed to synthesize biogenic Silver nanoparticles (AgNPs) using Panchgavya and evaluated antimicrobial activity against bacteria isolated from calf diarrhea samples. Fecal samples were collected from nine diarrheic calves and primarily tested for the presence of dominant bacteria using bacteriological examination. The predominant bacteria present in pooled calf diarrhea sample were characterized by 16s rRNA sequencing and identified as Escherichia coli and Proteus merabilis. The biogenic synthesis of AgNPs was initially observed through visual color change of silver nitrate solution from yellow to reddish brown and further confirmed by Surface Plasmonic Resonance band at 410 nm using UV-visible spectroscopy. The hydrodynamic particle size and zeta potential of synthesized AgNPs as observed by Dynamic Light Scattering spectroscopy was 169 nm and -56.0mV respectively indicating that AgNPs are highly stable. The stability of AgNPs was due to the presence of capping agents such as phenols, proteins, aromatic amines, free hydroxyl groups, alkene and amide groups derived from Panchgavya distillate which was established by Fourier Transform Infrared Spectroscopy. X-ray Diffraction study revealed the crystalline nature of AgNPs. Morphology and size of AgNPs were determined by Scanning Electron Microscopy. Shape of AgNPs was observed spherical with average size of 49nm. Panchgavya mediated biogenic AgNPs demonstrated antibacterial properties against Proteus mirabilis and E.coli bacteria isolated from calf diarrhea as observed by well diffusion assay, Minimal Inhibition Concentration (MIC) and Minimum Bactericidal Concentration (MBC) tests. The zone of inhibition for 100 μg and 50 μg of AgNPs against E.coli and Proteus mirabilis was 13 & 9 mm and 14 & 11 mm respectively. The MIC and MBC of AgNPs against Escherichia coli and Proteus mirabilis were 156.25 and 78.125 μg/ml respectively. These in vitro results indicate that the biogenic AgNPs present antimicrobial activity against E.coli and Proteus mirabilis of calf diarrheal origin.
