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ThesisItem Open Access Electroencephalographic and electrocardiographic studies on propofol anaesthesia in sheep(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2011-06) Raj Kumari; Das, Arup KumarIn this study electroencephalographic and electrocardiographic changes during surgical interventions {viz., placement of central venous catheters (Group-B), docking/ tail amputation (Group-C), bilateral orchiectomy (Group-D) and no surgical stimuli as control (Group-A)} with propofol anaesthesia were assessed in twenty-four (n=24) apparently healthy male sheep. For surgical operations each sheep of this study was premedicated with glycopyrrolate (@ 0.02 mg/kg body weight, IM) and, five minutes after, anaesthesia was induced with propofol (@ 4.00 mg/kg body weight, IV). The parameters in this study included clinical, cardiopulmonary, haematobiochemical and electrocardiographic parameters. Similarly, qualitative electroencephalographic evaluation was done through spectral analysis to generate total power, SEF-90, MF and PPF over 0-30 frequency band width while relative power of different bandwidths viz., , , and was assessed. Rectal temperature and respiration rate has not shown any significant variability in any of the groups during the entire course of experiment. Haemoglobin concentration reduced and this extent was higher in control group than central venous catheterization, docking and castration groups. PCV reduced in all the groups. TEC depicted reduction in control group; however, this fall was not apparent in other groups. Blood glucose decreased initially followed by an increase in three groups after propofol induction while the castration group exhibited a non-significant increase from very beginning. Total serum protein declined in all the groups. A sudden increase in serum triglyceride (TG) was observed at one minute post-propofol induction in all the groups, which later on declined. Propofol has not been able to induce any significant changes in P-wave, QRS wave and T-wave amplitude as exhibited in control group. However, P-wave amplitude increased significantly in animals performed with central venous catheterization. The T-wave amplitude increased nonsignificantly in CVC group while decrease in docking and castration groups. P-wave and T-wave duration has not exhibited any significant difference throughout the course of experiment. However, QRS wave duration decreased in control group and no significant changes were observed in surgical groups. PR segment length reduced in castration group only and none of the other groups exhibited any significant changes. ST segment length exhibited significant increase in control group, non-significant increase in CVC while exhibiting significant fall in castration and docking groups. RR interval exhibited reduction in all the four groups, non-significantly in control while significantly in the three surgical groups. Heart Rate increased in all the four groups. EEG analysis showed an increase in total power, median frequency, theta frequency, peak power in all group, and after a transient increase reduction in spectral edge frequency and alpha frequency. However delta frequency showed an increase after a transient decrease and a marked decrease in beta frequency after propofol injection. It was concluded that propofol to some extent possess analgesic effect and may be useful in husbandry practices/ day case surgery. However, evaluation of analgesic effects of propofol needs further investigation.ThesisItem Open Access Isolation, propagation and characterization of bone marrow derived mesenchymal stem cells and their differentiation into neurocytes in dogs(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-07) Verma, Rakesh Kumar; Kandpal, ManjulThe adult stem cell therapy is a blooming area of clinical research and relevance. The therapeutic benefits of mesenchymal stem cells in veterinary regenerative medicine are numerous as they are in human medicine. The mesenchymal stem cells in canines can be potentially used for cell based therapy to regenerate damaged or lost neuronal cells. The leitmotif of this study was differentiating cBM-MSCs into neurocytes in vitro. A healthy nondescript male dog of 3 years of age and 24kg weight was used for this study. The bone marrow aspirate was aseptically collected from ileac crest of the dog after administration of general anaesthesia by 2.5% thiopentone sodium till effect and atropine sulphate and diazepam as pre-anaesthetics. The bone marrow aspirate was processed in laboratory within 4 hours of its collection. Mesenchymal stem cells were isolated by density gradient centrifugation technique and cells were seeded in T-25 tissue culture flasks. After attainment of 80-90% confluence, first passage was performed by using Trypin-EDTA to expand the cell population. Cells were passaged till 4 passages and then grown in neurogenic differentiation media for 21 days in CO2 incubator at 37°C and 5% CO2. The morphology of cells was transitioned from spindle elongated shape to ring or net like shape. The cells were characterized at regular intervals by staining with the Nissl body stain after 21 days with positive result which coloured the nuclei blue-violet. The RT-PCR for the genes NSE and NFM revealed bands on days 21 confirming the differentiation process. The sample on gelatin scaffold and on pellet when subjected to scanning electron microscopy revealed net shape cells with cell aggregates. On the basis of this study it was concluded that MSCs possess potential to transdifferentiate to neurocytes when cultured in neurogenic media in vitro and the identification techniques are successful. The meticulous exploitation of MSCs in tissue engineering and regenerative therapy in veterinary medicine is the need of the hour.ThesisItem Open Access Isolation, expansion and characterization of bone marrow derived mesenchymal stem cells and their differentiation into hepatocytes in canines(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-07) Shaista Khanum; Kandpal, ManjulMesenchymal stem cells (MSCs), as adult stem cells (ASCs) able to divide into a variety of different cells, are of utmost importance for stem cell research. Mesenchymal stem cells (MSC) isolated from bone marrow and differentiated into hepatocyte-like cells have increasingly gained attention for clinical cell therapy of liver diseases because of their high regenerative capacity. The main focus of current study was on canine bone marrow derived mesenchymal stem cells and their differentiation towards hepatic lineage in vitro. The bone marrow was aspirated from iliac crest of young non-descript male dog under xylazine and ketamine anesthesia. The mononuclear cells along with mesenchymal stem cells were harvested by density gradient centrifugation. The mesenchymal stem cells were isolated on the basis of their plastic adherence property. The cells were seeded in tissue culture flasks and allowed to grow in monolayer till the attainment of 80-90% of confluency. At this confluency passaging was done using trypsin-EDTA and cultured with two different densities of 16 cells/cm2 and 4000 cell/cm2 in T-25 flasks. The MSCs cultured with initial seeding density of 4000 cell/cm2 showed higher yield up to 10 passages with faster expansion and followed standard growth kinetic curve. Thus, the fourth passage of these MSCs were made to differentiate into hepatocytes in stepwise manner including induction, differentiation and maturation steps. The differentiation was done by using growth factors HGF, bFGF and nicotinamide for induction, HGF, ITS and Dexamethasone for differentiation and OSM, ITS and dexamethasone for maturation. The cells were cultured for 30 days in an incubator maintained at 5% CO2 and 37oC.The morphology of cells was observed at regular intervals and cells were characterized by PAS staining, ICG uptake, gene expression analysis and SEM. The cells demonstrated positive PAS staining by giving pink to purple red colour. The cells also affirmed the ICG uptake by manifestation of dark green colour nuclei. The RT-PCR analysis revealed the expressions of hepatocyte specific markers alfa fetoprotein (AFP), albumin (ALB), tyrosine aminotransferase (TAT) and alpha-1 antitrypsin (_1-AT) in differentiated cells. The cells when subjected to scanning electron microscopy also revealed hepatocyte like morphology. On the basis of this study it was concluded that canine bone marrow derived mesenchymal stem cells were expanded successfully up to 10 passages with optimum initial seeding density and possessed potential for differentiating into hepatocyte-like cells in vitro. The exploitation of this in vitro harvested hepatocyte like cells may prove an effective therapy for liver diseases.ThesisItem Open Access Isolation, propagation and characterization of bone marrow derived mesenchymal stem cells and their differentiation into cardiac myocytes in canines(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-07) Khalid Mubarik; Kandpal, ManjulMesenchymal stem cells are valuable source of multipotent cells which have the ability to differentiate into various types of tissue cells and contribute to the regeneration of a variety of mesenchymal tissues including bone, cartilage, muscle, and adipose. Bone marrow-derived mesenchymal stem cell (MSC)-based therapy is an emerging approach for the treatment of both acute and chronic heart failure. The aim the current study was to focus on canine mesenchymal stem cells and to analyse their differentiation capacity into cardiomyocyte phenotype in vitro. An adult mongrel dog of 5 years of age weighing 25 kgs was used in this study. Bone marrow was aspirated from iliac crest after anaesthesia using xylazine and ketamine. Mesenchymal stem cells were harvested using density gradient centrifugation from the mononuclear cells of bone marrow. The cells were seeded in tissue culture flasks and allowed to grow in monolayer till the attainment of 80-90% of confluency. At this confluency passaging was done using trypsin-EDTA. The cells were subcultured upto four passages. These cells were characterized at their baseline for morphology, culture behavior and also by scanning electron microscopy. At passage three, the cells were exposed to medium containing 5-Azacytidine (10µl/ml) to induce differentiation into cardiac myocyte. The media was changed into media without 5-Azacytidine after 24 hours. The cells were cultured for 21 days in an incubator maintained at 5% CO2 and 37oC. The differentiation of these cells was then analysed using cellular morphology, gene expression and scanning electron microscopy. PCR was carried out for expression of genes Nkx 2.5, Cardiac troponin-T (cTnT), β-Cardiac myosin in differentiated cells. SEM was also done for analyzing the morphology of differentiated cells. Canine MSCs demonstrated morphological changes towards cardiomyocyte like phenotype during differentiation. Cells assumed a stick-like morphology and were connecting with adjoining cells forming myotubelike structures. The cells demonstrated increased expression of cardiac specific markers. On the basis of results obtained in this study it was concluded that Mesenchymal stem cells possess potential for differentiating into cardiac myocyte like cells in vitro. The exploitation of these in vitro harvested cardiac myocyte like cells may prove an effective therapy for cardiac repair under various pathological conditions.ThesisItem Open Access Isolation, expansion and characterization of bone marrow derived mesenchymal stem cells and their differentiation into skeletal myocytes in canines(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-06) Sandhu, Ramanpreet Singh; Jadon, Narendra SinghThe adult stem cell therapy is a blooming area of clinical research and relevance. The therapeutic benefits of mesenchymal stem cells in veterinary regenerative medicine are numerous as they are in human medicine. The characterization of canine derived MSCs is poorly mentioned in literature. The mesenchymal stem cells in canines can be potentially used for cell based therapy to regenerate damaged or lost muscle cells. The motive behind this study was fixed on the differentiation potential of canine mesenchymal stem cells into skeletal myocytes in vitro. An adult male mongrel dog of 3 years of age and 24kg weight was used for this study. The bone marrow aspirate was aseptically collected from ileac crest of the dog after administration of general anaesthesia by 2.5% thiopentone sodium till effect and atropine sulphate and diazepam as pre-anaesthetics. The bone marrow aspirate was processed in laboratory within 4 hours of its collection. Mesenchymal stem cells were isolated by density gradient centrifugation technique and cells were seeded in T-25 tissue culture flasks. After attainment of 80-90% confluence, first passage was performed by using Trypin-EDTA to expand the cell population. Cells were passaged till 4 passages and then grown in skeletal myogenic differentiation media for 21 days in CO2 incubator at 37 ° C and 5% CO2. The morphology of cells was observed at regular intervals and the cells were characterized by use of Hoechst 33342 stain after 21 days with positive result which coloured the nuclei blue. The RT-PCR for the genes Myogenin and Myo-D revealed bands on days 7 and 14 confirming the differentiation process as early differentiation factors. The striations were observed when the sample on gelatin scaffold was subjected to scanning electron microscopy and close association of parallel arranged cells with striations when the pellet was subjected to electron microscopy. On the basis of this study it was concluded that MSCs possess potential to trans differentiate to skeletal myocytes when cultured in skeletal myogenic media in vitro and the identification techniques are successful. The meticulous exploitation of MSCs in tissue engineering and regenerative therapy in veterinary medicine is the need of the hour.ThesisItem Open Access Clinical study on canine cystic endometrial hyperplasia complex(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-06) Pushpa; Das, Arup KumarThe objectives of the study were to evaluate the effect of CEH-P on clinical, haematobiochemical and hormonal profile of dogs and changes after OH. Cystic endometrial hyperplasia is a pathological complex which incorporates conditions like cystic ovary, endometrial hyperplasia and accumulation of mucoid material in the lumen of the uterus which might be invaded by opportunistic bacteria and may gradually proceed to pyometra. This study was conducted on 18 female dogs and divided in to two groups in group A 12 female dogs with symptoms of endometrial hyperplasia complex were diagnosed and in group B rest of 6 female dogs were operated for elective ovarianhysterectomy and such animals were kept as control group. Clinical parameters, haematobiochemical parameters, hormonal profile and histo-pathological examination were observed for all the 18 animals just before the operative procedure and after 45 days of the ovarian-hysterectomy. The hormonal profile (estradiol, progesterone, LH and FSH) of the animals revealed that significant differences in the levels of steroid hormones mainly in progesterone and estrogen in female dogs suffering from cystic endometrial hyperplasia complex as compared to normal female dogs and most of the animal suffering from CEHP complex were having either history of pervious hormonal treatment or they were not bred several years before showing symptoms so there is disturbances in level of steroidal hormone mostly progesterone, which causes increases sensitivity of animals for development of pyometra and CEH. Levels of these hormones after ovarian hysterectomy procedure reduced significantly (109.67 pg/ml, 4.25 ng/ml 17.76pg/ml to, 0.17ng/ml estrogen and progesterone pre and post operatively respectively). This study concludes that in this pathological condition of the uterus the levels of steroid hormones increases, change in their receptors and exogenous administration of theses hormone may play an important role in pathogenesis of CEH-P complex. The main hormone responsible for CEH-P Complex is Progesterone. FSH and LH are not play important role in development of CEH-P complex. The ovariohisterectomuy is choice of treatment for pyometra in female dogs.ThesisItem Open Access Isolation, propagation and characterization of bone marrow derived mesenchymal stem cells and their differentiation into chondrocytes in dogs(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-06) Joshi, Pratibha; Jadon, Narendra SinghThe intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells are contemplated as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The leitmotif of this study was differentiating cBM-MSCs into chondrocytes in vitro. The aspiration of bone marrow was done aseptically from ileac crest of the mongrel dog. The procedure was done after administration of general anaesthesia. The aspirate from bone marrow was processed within half an hour of its collection. Pellet of MSCs along with mononuclear cells were isolated by density gradient centrifugation technique. Mesenchymal stem cells were isolated on the basis of plastic adherence and elongated spindle –shape. At 80-90% confluency, cells were passaged using Trypin-EDTA to amplify the cell population. Passaging of cells were done upto 4 passages and then MSCs were grown in chondrogenic differentiation media in CO2 incubator at 37 ° C and 5% CO2 for 21 days. The morphology of cells was transitioned from spindle elongated shape to round/ spherical shape. The cells were characterized at regular intervals by staining with Toluidine blue at 7th and 21st days with positive result which coloured the extracellular matrix purple. The RT-PCR for the genes SOX9 and COMP at 10th day supported with Type II collagen and Aggrecan at 22nd day revealed bands confirming the differentiation process. The sample on gelatin scaffold and on pellet when subjected to scanning electron microscopy revealed round shape cells with cell aggregates. It was concluded with the support of this study that MSCs possess potential to differentiate into chondrocytes when cultured in chondrogenic medium in vitro and techniques for identification are affluent. It had nevertheless paved the way for the establishment of regenerative and gene therapy for repair and regeneration of damaged and diseased tissue.ThesisItem Open Access Immunity related gene expression during progression and regression of canine transmissible venereal tumour(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-06) Tiwari, Kanika; Das, Arup KumarThe present study was conducted on clinical cases of twenty four dogs of either sex or age affected with naturally occurring canine transmissible venereal tumour (CTVT) to evaluate the gene expression during progression and regression of tumour and to evaluate the T cell response toward mitogen Concanavalin A. Animals were randomly divided into four groups consisting of 6 dogs each. Group 1 consists of TVT affected dogs in which vincristine sulphate were not administered. Group-2 consists of normal dogs in which vincristine sulphate were administered. Group-3 consists of TVT affected dogs in which vincristine sulphate were administered. Control group consists of normal dogs in which vincristine sulphate were not administered. The assessment were made on the basis of clinical examination, gross regression after chemotherapy, haemato-biochemical, lymphocyte proliferation assay and expression of gene were studied before and after administration of vincristine sulphate in CTVT affected dogs. On the basis of these parameters, it is concluded that transforming growth factor beta 1 (TGF-β1) was secreted by CTVT cells during progression phase which suppressed the lymphokine activated killer (LAK) cytotoxicity, which is suppressed by CTVT cells secreted IL-6 during the regression phase of the tumour. T- lymphocytes are also associated with the regression phase of CTVT suggested that tumour immunity also plays a major role in tumour regression.ThesisItem Open Access Comparative evaluation of external skeletal fixator, hybrid tie-in fixator and intramedullary Titanium pinning for repair of induced humerus fracture in fowl (Gallus gallus)(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2016-08) Mir Tufail Mohd.; Das, Arup Kumar