Isolation, Characterization Of Chondroitin Sulphate And Its Efficacy In Osteoarthritis
Material type: Book ; Format:
; Literary form:
Publisher: 2012 Dissertation note: Chondroitin sulphate (CS) and Glucosamine sulphate (GS) are two main components of articular cartilage. It is believed that these molecules slow down wear and tear of cartilage. Moreover, if administered exogenously as drugs, these may initiate synthesizing capacity of cartilage. Among these, GS promotes the formation and repair of cartilage, whereas CS promotes elasticity and prevent cartilage breakdown by inhibiting degradative enzymes. Concurrent use of both structural units of cartilage as drugs in osteoarthritis (OA) may lessen the progression of disease.
The present study was conducted to elucidate the chicken keel cartilage as an alternate and potential source for this endogenous component that may be used exogenously to repair or prevent damage to joints. Chicken keel cartilages were collected from healthy broilers. CS was extracted using MgCl2 solution (3M), dialyzed and digested with papain. The extracted material was purified by ethanol precipitation, centrifugation and then freeze dried. Proximate analysis of semi-purified polysaccharides revealed the presence of carbohydrates (65.49±0.10), crude protein (12.82±0.26), ash (11.12±.56), moisture (9.88±0.32) and fat (0.69±0.14). Fiber contents were found to be nil in the processed samples. Dimethylmethylene blue binding (DMMB) assay was performed for determination of percent contents of CS in extracted semi-purified samples and mean concentration was found to be 70.77±2.35. Semi-purified polysaccharides were further characterized by FTIR (Fourier Transform Infrared Spectrometer) technique and characteristic Peaks of CS molecules were recorded at 854, 854 and 853 cm-1 and then compared with spectrum of standard CS. Protein content being a major impurity in extracted samples was determined by Bradford method quantitatively (4.64±0.29). Two protein impurities having 77.8 and 50.5 kDa molecular weights were revealed by SDS-PAGE.
Efficacy of semi-purified CS from chicken keel cartilage, standard CS from shark source and GS, alone and in combination in experimental OA rat model was evaluated. To develop OA similar to spontaneous OA, 10mg papain/0.5mL (Sigma, Cat # P 3125) in buffered solution of 0.05 M sodium acetate pH 4.5 was injected intra-articularly in each right knee joint of fifty five albino rats (pre-anesthetized with anesthetic ether). Ten rats (n= 10) were injected with 0.5mL of normal saline (0.9%) in right knee joint that served as control group. Then from fifty five papain injected rats, twenty five were divided into five groups (n=5) for development and assessment of OA model (OA groups). Progression of disease was monitored by clinical scores, histopathological scores and concentration of CTX-II as biomarker in sera samples of experimental rats by ELISA using a commercial kit (serum preclinical CartiLaps ® ELISA kit) for control and OA groups (n=5) on day 0 (control group) and days 1st, 7th, 14th, 21st and 28th post papain injection (OA groups). Highest mean clinical score (10.38±1.1) was observed on 1st day and least on 28th day post papain injection i.e. 5.00±.34. Highest mean histopathological score and CTX-II concentration was recorded on 28th day i.e. 12.82±1.64 and 36.82±3.81. Values of clinical scores, histopathological scores and CTX-II concentration reached to maximum on 21st day and then sustained thereon. Second phase of experiment is comprised of evaluating and comparing the efficacy of extracted CS samples (chicken keel cartilages), standard CS (shark source) alone and in combination with GS. For this purpose, remaining five rats out of ten injected with normal saline intra-articularly served as control groups along with treated and non treated groups of experimental rats. Remaining thirty OA induced rats were divided into six groups (five rats /group). Group 1 (n=5) called non treated group received only placebo till 60th day and served as negative control group.
Treated Group 2 received GS alone, Group 3 CS (standard) and Group 4 were given extracted CS. Group 5 was treated with combination of GS plus CS (standard) and Group 6 with GS plus CS (sample). Doses of glycosaminoglycans (GAGs) were administered as 1.2g/kg/day CS and 1.5g/kg/day GS alone and in combinations. Drugs were offered early in the morning in bolus form with feed (10g) after overnight fasting while non-treated group received only placebo (without any drug).
Anti-arthritis activities of CS standard and extracted alone and in combination with GS were assessed clinically, analyzed statistically by using one way ANOVA. Level of significance (P<0.05) was recorded by using Duncan's Multiple Range (DMR) Post hoc Test. Mean scores of clinical, histopathology and CTX-II concentrations observed at 60th day in control rats (without OA) were 0.00, 0.00 and 2.55, respectively. OA induced untreated group showed mean score for clinical signs, histopathological scores and CTX-II concentrations 4.15, 12.24 and 36.70 and GS treated group 3.19, 3.96 and 6.12 at 60th day of treatment, respectively. For CS (standard), mean scores of clinical signs, histopathological lesions and CTX-II concentrations were recorded as 2.64, 2.44 and 4.48 and for CS (extracted) were 2.26, 2.28 and 4.40 in sera correspondingly at 60th day of treatment. The lowest mean values of clinical signs, histopathology and CTX-II concentrations in sera of treated group with standard CS plus GS were found to be 0.94, 0.94 and 2.62 followed by extracted CS plus GS treated groups 01.05, 1.27 and 2.74, respectively. Clinical, histopathological scores and CTX-II concentrations in group of rats treated with combinations were found to reverse the diseased condition after 60th days of treatment as the values were close to that of normal rats and far away from OA rats. It is concluded that extracted CS from poultry has comparable efficacy with CS standard from shark source alone and in combination with GS. Poultry by-product (keel cartilage) is found to be an alternate and cheap source for CS (chondroprotective agent) as compare to expensive, less available and religiously prohibited source for Islamic countries particularly.
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Comparison Of Commercial Triladyl Extender With A Tris-Citric-Egg-Yolk (TCEY) Extender On Post-Thaw Semen Quality Of Nili Ravi Buffalo
Material type: Book ; Literary form:
Publisher: 2016 Dissertation note: Cryopreservation of semen is the most important step for its usage in artificial insemination. Freezing of semen leads to a remarkable reduction in post-thaw semen quality. Therefore, selection of a better semen extender has always been considered priority that could serve as a good cryoprotectant.. Our semen production units (SPUs) have been using Tris based egg yolk semen extender since long time. Some modern SPUs like CEBG are using commercially available semen extenders for better post-thaw semen quality.
After collection pooled semen divided into two equal aliquots in separate sterilized test tubes and kept in water bath at 37 ºC. Semen was diluted with each of extender (TCEY and Triladyl) on the basis of sperm concentration (40x106sperm/ ml). Diluted semen was placed bottles and placed in safety cabinet cooled to 4 ºC over and equilibrated for 4 hrs. After equilibration semen was filled in 0.5 ml French straws (20x106sperm/ 0.5 ml). All semen straws placed in automatic freezer 4cm above liquid nitrogen surface in vapors for 10 minutes. Liquid Nitrogen vapors used in automatic programmable freezer to reduce temperature from 4 ºC to -180 ºC and then plunged into liquid nitrogen -196 ºC for freezing and was stored until analyzed. The experiment was repeated for seven times (replicates = 07)
CASA sperm motility parameter and kinematics were analyzed at Center of Excellence for Bovine Genetics (CEBG) Renala khurd District Okara. For further analysis frozen semen straws were brought to the Department of Theriogenology UVAS, Lahore. Effects of Triladyl and TCEY on post-thaw semen quality of the Nili Ravi buffalo semen were compared.
In Triladyl group, significantly (P<0.05) higher post-thaw motility (PTM %), Plasma membrane integrity (PMI, %),) DNA integrity (%), Live percentage was found. However, no significant (P<0.05) difference was found regarding NAR results between both groups. Sperm abnormalities were found significantly lower in Triladyl group as compared to TCEY group.
In overall assessment regarding and post-thaw CASA motility parameters, CASA motility, (PROG %), rapid (RAP %), medium (MED%), and slow (Slow, %) and sperm motility kinematics (VAP μm/sec), (VSL μm/sec), (VCL μm/sec), (ALH μm), (BCF HZ), (STR%) and (LIN%) Triladyl was found better than TCEY.
This was concluded that use of commercial semen extender Triladyl resulted in significantly better post-thaw semen quality as compared to Tris citric egg yolk (TCEY) extender.
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Characterization And Antibiotic Resistance Profile Of Listeria Monocytogenes Isolated From Fish And Broiler Meat
Material type: Book ; Literary form:
Publisher: 2017 Dissertation note: Gram positive rod shaped, non-spore former Listeria monocytogenes is the major food-borne pathogens for humans and animals. It can cause serious foodborne infection. The bacterium is saprophyte and can grow on wide range of temperature (0-45°C). Due to this it contaminates different food products. Consumption of this contaminated food products can cause serious problems in neonates, pregnant women and immunocompromised peoples. Its signs may develop between day one to three months after ingestion of the organism. The neonates can develop septicemia, respiratory diseases and meningitis. The pregnant woman may develop influenza like symptoms, or keep an asymptomatic infection that ends in abortion, premature birth or sepsis in the newborn. Healthy people hardly develop clinical signs but a febrile gastroenteritis syndrome has been reported. No doubt this disease is associated with unhygienic food consumption and is characterized by fever, nausea, diarrhea, headache, abdominal pain and sometime myalgia. These symptoms may be resolved in one to three days Listeria monocytogenes was isolated by conventional methods and suspected colonies were identified by Gram staining and biochemical tests catalase and oxidase test. The DNA was extracted from isolated colonies by 10% chelex method. The isolated strains were confirmed through PCR by targeting prfA gene of 479bp. Antibiotic resistance were also checked for confirmed isolates. A total of 160 (Fish meat n=80, Broiler meat n=80) samples were taken for the present study for screening Listeria monocytogenes. The bacterium was found in 19/80 (23.75%) samples of Fish and 5/80 (6.25%) of broiler meat samples through PCR detection.
Later the confirmed isolates were tested to check the resistance profile of the bacterium to different commonly available antibiotics. For this 24hrs old culture were used. Three to five colonies were picked by sterile loop and transfer to test tube containing 10ml normal saline. To check the turbidity the tube was compared with 0.5 Macfarland standard. Then by sterile cotton swab the bacterium was spread on Mueller Hinton agar. Antibiotics were placed by sterile forceps on agar. The plates were incubated at 37°C for 24hrs. Zones of inhibition were measured in mm by the help of ruler and then compared with staphlycoccus aureus break points in CLSI. The susceptibility result shows that the bacterium was resistant to gentamicin. Mostly L. monocytogenes isolates were susceptible to antibiotics used in this study. This study suggested ampicillin as drug of choice for treatment of listeriosis. Preventive measures should be adopted to avoid the risk of the disease.
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