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ThesisItem Open Access GENOTYPING AND PROTEIN PROFILING OF MILK β-CASEIN VARIANTS (A1 and A2) IN INDIAN BREEDS OF MILCH CATTLE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2018-03) SRINIVAS, B; ASWANI KUMAR, K(MAJOR); PADMAJA, K; PUNYA KUMARI, BABSTRACT: The exotic and cross bred cattle milk contain A1 β-casein variant, whereas indigenous cattle milk contains A2 β-casein variant. They have structural diversity at 67th amino acid residue proline in A2 which is replaced by histidine in A1 during course of evolution. The histidine at 67th amino acid residue generates a proteolytic cleavage site in A1 leading to the production of betacasomorphin-7 (BCM-7) that is known to be a risk factor for several human diseases. The serious health concern associated with A1 milk consumption endures an urgent need to select cattle breeds with higher genotypic and allelic frequencies of A2 and conserve indigenous cattle breeds through breeding programmes. In this context, the present study was undertaken to screen seventy animals belonging to five indigenous cattle breeds of India viz., Deoni, Sahiwal, Ongole, Punganur, Malnad Gidda and Holstein Friesan cross (twelve from each breed and ten from Deoni) to characterize β-casein variants (A1 and A2) geotypically by PCR-RFLP with TaqI restriction enzyme and also to study milk β-casein variants (A1 and A2) by IEF. PCR-RFLP with Taq1 restriction enzyme yielded two genotypes as A1A2 (251 and 213 bp) and A2A2 (251 bp) in all indigenous cattle breeds studied, whereas only heterozygous (A1A2) genotype was found in the HF crossbreds. The genotypic frequency of A1A2 heterozygous variant of exon 7 of CSN2 gene was the highest for HF cross (1.0) followed by Sahiwal (0.75), Deoni (0.58), Malnad Gidda (0.40), Punganur (0.17) and Ongole (0.083). Whereas, for A2A2 homozygous variant was the highest for Ongole (0.92) followed by Punganur (0.83), Malnad Gidda (0.60), Deoni (0.42) and Sahiwal (0.25). The A1 allelic frequency of exon7 of CSN2 gene was the highest in HF cross (0.50) followed by Sahiwal (0.38), Deoni (0.29), Malnad Gidda (0.20), Punganur (0.083) and Ongole (0.042), whereas the highest A2 allelic frequency was observed in Ongole (0.96) followed by Punganur (0.92), Malnad Gidda (0.80), Deoni (0.71), Sahiwal (0.63) and HF cross (0.50). The IEF gel electrophoresis pattern of milk β-casein protein showed two distinct bands corresponding to A1 and A2 β-casein protein variants at pI values 5.22 and 5.14 respectively and this polymorphism correlated positively with the results of PCR-RFLP with TaqI restriction enzyme. The present study unveils the existing fact that there is incorporation of exotic germplasm in native cattle breeds to varying degrees in different breeds and emphasizes an immediate need for genotypic screening of bulls for β-casein gene variant A2 for documentation and further measures to improve the indigenous breeds with appropriate breeding strategies.ThesisItem Open Access A COMPARATIVE STUDY ON HAEMATOLOGICAL AND BIOCHEMICAL PARAMETERS IN NORMAL CYCLIC, REPEAT BREEDING AND ANOESTRUM CROSSBRED MURRAH BUFFALOES(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2018-02) ANITA, K; PADMAJA, K(MAJOR); PRASADA RAO, T; PUNYA KUMARI, BABSTRACT: The present study was planned to assess the levels of haematological parameters and biochemical constituents in normal cyclic, repeat breeding and anoestrum crossbred Murrah buffaloes in drought area Proddatur in YSR Kadapa district of Andhra Pradesh. For this study a total of 60 blood samples were collected from each 20 healthy, 20 repeat breeding and 20 anoestrum buffaloes brought to the Department of Veterinary Clinical Complex, College of Veterinary Science, Proddatur and from various Veterinary dispensaries and Veterinary camps conducted in and around Proddatur. The variations in circulating levels of biochemical constituents and haematological parameters have been recorded in normal cyclic, repeat breeding and anoestrum crossbred Murrah buffaloes. The results showed significant decrease in glucose, cholesterol, total protein, albumin, globulin levels and significant increase in triglycerides and blood urea nitrogen in repeat breeding and anoestrum compared to normal cyclic crossbred Murrah buffaloes. Alkaline phosphatase and aspartate aminotransferase activities significantly elevated in repeat breeding and decreased in anoestrum compared to normal cyclic crossbred Murrah buffaloes. Whereas, alanine aminotransferase activity significantly decreased both in repeat breeding and anoestrum compared to normal cyclic crossbred Murrah buffaloes. Significant reduction was observed in calcium and phosphorous levels in repeat breeding and anoestrum compared to normal cyclic crossbred Murrah buffaloes. Alterations observed in sodium, potassium and chloride levels among the groups were marginal. The haematological parameters viz., haemoglobin, total erythrocyte count, total leucocyte count, packed cell volume, platelet count, monocytes, eosinophils and neutrophils were significantly decreased in repeat breeding and anoestrum conditions compared to normal cyclic animals. Whereas, lymphocytes and basophils were significantly increased in repeat breeding and anoestrum conditions compared to normal cyclic animals. In repeat breeding and anoestrum conditions increased malondialdehyde and decreased superoxide dismutase and catalase activities noticed compared to normal cyclic crossbred Murrah buffaloes.ThesisItem Open Access ISOLATION AND PURIFICATION OF GLUTATHIONE-S-TRANSFERASE (GST) AND ITS ROLE ON OXIDATIVE STRESS IN HEPATIC TISSUES OF MAREK’S DISEASE AFFECTED CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-01) RAJIV KISHORE, K.V.; PADMAJA, K(MAJOR); ESWARA PRASAD, P; PUNYA KUMARI, BABSTRACT: A study was conducted to isolate and purify Glutathione-S-transferase (GST) and to determine its role on oxidative stress in liver tissues of Marek’s disease (MD) affected chicken. Ten healthy and MD suspected layer birds of 35-40 weeks age for each group were procured from poultry farms in Chittoor district of A.P. The postmortem examination of MD affected birds revealed enlarged liver with discrete grayish nodules of various sizes. Histopathological examination of liver revealed extensive proliferation and infiltration of pleomorphic cells. The purified DNA samples from liver tissue of MD suspected birds were further used for PCR analysis which was found to be positive yielding a 314 bp product indicating the presence of two copies of 132 bp tandem repeat and a 50 bp primer sequence. The liver samples positive for MD were used for further analysis. The results showed a significant increase of TBARS (179.82%) in liver of MD affected chicken compared to control. Glutathione (GSH) levels were significantly reduced (52%) in liver of MD affected chicken compared to control. A significant decrease in SOD (32%), catalase (43%), GPx (55%) and GST (51%) activities were observed in MD affected chicken compared to control. The GST enzyme was isolated, purified and characterized from liver tissue of both healthy and MD affected layer chicken using glutathione agarose affinity chromatography. The enzyme was purified to 2.38 fold with a yield of 32.27% in healthy chicken whereas in MD affected chicken it was purified to 4.54 fold with a yield of 41.98%. The analysis of active fractions using SDS-PAGE showed GST-α expression with a molecular weight of 25.6kDa in both healthy and MD affected liver. In addition GST-π was expressed in MD affected chicken with a MW of 27 kDa. These results confirm that Marek’s disease in chicken induces oxidative stress which in turn leads to alterations in antioxidant enzymes. The oxidative stress induced in liver is responsible for GST π expression as a defense mechanism in MD affected chicken.ThesisItem Open Access EFFECT OF METHANOLIC EXTRACT OF Caesalpinia sappan L. ON ANTIOXIDANT ACTIVITY, GROWTH PERFORMANCE AND CARCASS CHARACTERISTICS IN BROILER CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2015-06) GIRINATH, Ch; ESWARA PRASAD, P(MAJOR); ASWANI KUMAR, K; ADILAXMAMMA, KABSTRACT : Oxidative stress in broiler chicken can result in damage to biomolecules, cells and tissues which thereby can negatively affect antioxidant system and result in poor growth rate and production of the broiler chicken. A trial was conducted during the summer months of May-June of 2014 when the temperature and relative humidity were in the range of 38 to 40ºC and 46 per cent respectively to study the antioxidant and growth promoting effect of methanolic extract of Caesalpinia sappan in broilers. One hundred day-old broiler chicks were randomly assigned into five groups with 20 birds each. Control group I was given basal diet and treatment groups II, III and IV were supplemented with methanolic extract of Caesalpinia sappan stem bark @ 100, 300 and 500 ppm respectively and group V was administered with vitamin E @ 100 ppm as an antioxidant control through drinking water from day 0 to 8 weeks of age. Serum and tissue samples were collected at the end of 8th week of the experiment and preserved at -20ᵒC for analysis of tissue antioxidant profile (TBARS, reduced glutathione, GPx, SOD, catalase) and serum biochemical parameters like ALT, AST, CK, cholesterol and T3 activity. The performance parameters were recorded at weekly intervals. The results indicated that the mean values of TBARS levels were significantly decreased whereas the levels of reduced glutathione, GPx, SOD and catalase were significantly increased in liver and kidney tissues of treatment groups compared to control and their activity was comparable with that of vitamin E treated antioxidant control group indicating heat stress induced oxidative damage to the tissues. The serum concentrations of ALT, AST, CK and cholesterol were significantly lower in C. sappan treated group III and IV compared to control group. However, serum T3 levels showed no significant change among different groups. Growth performance did not differ among different treatment groups during the starter period. In the finisher phase and overall period, birds in group IV had higher feed intake, body weight gain and feed conversion ratio, followed by birds in group III when compared with control group, which showed the lowest performance. The dressing yields and abdominal fat percentages were not influenced by various dietary treatments, but group IV birds showed better dressing percentage compared to control group. The results of this study concluded that, supplementation of methanolic extract of Caesalpinia sappan improved antioxidant status and their performance in broiler chickens reared under high ambient temperature during summer months and it can be considered as a replacement for synthetic antioxidant in poultry diet in heat stress conditionsThesisItem Open Access CONJUGATION OF ANTIBIOTIC RESIDUES PREVALENT IN MILK WITH CARRIER PROTEIN AND RAISING OF POLYCLONAL ANTIBODIES AGAINST THEM(SRI VENKATESWARA VETERINARY UNIVERSITY , TIRUPATI – 517502. (A.P.) INDIA, 2012-08) SAMPATH KUMAR, B; ASHOK, VASILI (Major); ESWAR PRASAD, P; Krishnaiah, NABSTRACT : Antibiotics are the drugs used to kill or inhibit the growth of bacteria. Presence of antibiotic residues in milk has significant public health importance as they have adverse side effects on the health. The present study was undertaken to produce polyclonal antibodies against the most commonly used antibiotics in large animal practice so that a diagnostic test can be developed to screen milk samples for the presence of antibiotic residues. Ampicillin, ceftiofur, enrofloxacin and gentamycin were conjugated with carrier protein BSA by carbodiimide method in which EDC was used as a crosslinking agent. Successful conjugation was confirmed by SDS PAGE in which antibiotic conjugates revealed high molecular weight protein bands of 90kDa, 85kDa, 78kDa, 74kDa for gentamycin, ampicillin, ceftiofur and enrofloxacin respectively in contrast to 68kDa band of BSA. Sprague Dawley rats aged 7-8 weeks were immunized with the antibiotic-BSA conjugates by subcutaneous route according to the standard immunization schedule. A total of four blood samplings were done from each group (3 animals) as follows: first three samples serially at 15 days time interval after 1st immunization plus 1st booster, 2nd booster, 3rd booster and the 4th sampling one and half month after the third booster. The mean total protein content in the antisera of ampicillin, ceftiofur, enrofloxacin and gentamycin groups were 12±1.15 g/dL, 30±1.20 g/dL, 24±1.73 g/dL and 8.25±0.20 g/dL respectively and in the control group serum it was 6.66±0.01 g/dL. The mean albumin concentrations were 3.44±0.020 g/dL, 3.36±0.173 g/dL, 3.21±0.003 g/dL & 2.93±0.014 g/dL for ceftiofur, ampicillin, enrofloxacin and gentamycin groups respectively compared to 3.36±0.173 g/dL in the control group. The mean globulin content in the antisera of ampicillin, ceftiofur, enrofloxacin and gentamycin groups were 8.64±0.10 g/dL, 26.56±1.18 g/dL, 20.79±0.08 g/dL and 5.32±0.05 g/dL respectively and it was 3.30±0.04 g/dL in the control group. A/G ratios were less than the normal value (>1) in antisera of ampicillin, ceftiofur, enrofloxacin and gentamycin groups. Indirect ELISA was standardized with antibiotic-casein conjugate as coating antigen to detect antibody titres. The optimum antigen concentration was chosen as 20ng/ml (which was in the range of Maximum residue limits) and optimum antiserum dilution was chosen as 1/100 by checker board titration. The antibody titres in the test samples of 4 immunization cycles for each antibiotic were determined by indirect ELISA at various serum dilutions ranging from 1/100 to 1/6400. Cut off value of 0.3 was calculated from PNT base line constructed with M+3SD values of control serum samples. All the four antibiotic-BSA conjugates elicited positive immune response in rats. The immune response in BSA-ampicillin conjugate immunized rats was maximum with the highest antibody titre of 2.577 (OD450) followed by BSA-ceftiofur conjugate immunized rats, BSA-enrofloxacin conjugate immunized rats and BSA-gentamycin immunized rats with OD450 values of 2.072, 1.723, and 0.928 respectively at the antiserum dilution of 1/00. These polyclonal antibodies are the efficient tools for developing immunoassays to detect specific antibiotic residues in biological fluids like milk and urineThesisItem Open Access EFFICACY OF ALOIN MEDIATED PHYTOGENIC NANOSILVER PARTICLES IN EXPERIMENTAL MURINE MASTITIS(SRI VENKATESWARA VETERINARY UNIVERSITY , TIRUPATI – 517502. (A.P.) INDIA, 2013-02) VENKATA CHAITANYA KUMAR, T; ESWARA PRASAD, P (Major); PADMAJA, K; SREENIVASULU, DABSTRACT : An experimental study was conducted to evaluate the efficacy of aloin mediated phytogenic nanosilver particles in experimental murine mastitis. The nanoparticles were synthesized and the synthesized nanoparticles were characterized using UV-visible spectrophotometer, Scanning electron microscope (SEM), DLS (Dynamic light scaterring) Technique and FT-IR analysis and were also evaluated for antimicrobial activity and cytotoxic effects. A total of 40 female mice between 10-15 days of lactation were utilized for the study. The animals were divided into five groups of eight animals each. Group I served as lactating control, groups II to V were inoculated with 20μl of 24h broth culture of S.aureus containing 4.0 x 104 cfu/quarter under ketamine anaesthesia using 33g blunt hamilton needle. After 6h post inoculation, groups III and IV received 20μl of Aloin nano silver (ANS) through intramammary and intra peritoneal routes respectively. Group V received antibiotic Cefepime at a dose rate of 1mg/kg body weight through intra peritoneal route. After 18 h post treatment, serum C reactive protein (CRP) levels, weights of mammary glands, mammary bacterial loads, levels of TBARS, GSH, SOD and CAT, histopathology and ultrastructure studies were conducted. UV Visible absorption spectrum showed a maximum absorption around 200 nm for ANS. SEM images showed that ANS with cubical, rectangular, triangular and spherical morphology of size 287.5 to 293.2 nm. The results of FT-IR analysis indicated the involvement of hydroxyl, carboxyl, amine and nitrile groups in the synthesis of aloin mediated silver nanoparticles. The size of the particles as measured by DLS technique was 67.8 nm. The compound (ANS) showed an MIC of 21.8 ng/ml against Staphylococcus aureus and showed an in vitro spleenocyte viability of more than 80% at the highest concentration of 87.5 ppm per well. The compound was also found to be safe at the limit dose in acute oral toxicity conducted in rats. The bacterial load was significantly reduced in ANS (intramammary), ANS (intra peritoneal) and antibiotic group compared to mastitis control. The levels of C-Reactive Protein were significantly reduced in Aloin treatment groups III and IV compared with antibiotic group V. TBARS was not significantly reduced to the level of normal lactating group, however SOD and CAT levels were lowered by both routes of aloin nanosilver (ANS) treatments. In histopathological sections, significant reduction (98%) in PMN infiltration was observed with ANS than antibiotic (50%). Ultrastructure of mammary gland from aloin treatment groups III and IV indicated moderate progress in the architecture of the secretory epithelial cells showing reconstituting mitochondria and normal inter alveolar septa.ThesisItem Open Access COMPARATIVE EVALUATION OF WOUND HEALING PROPERTIES OF NANOSILVER AND BOSWELLIA OVALIFOLIOLATA MEDIATED NANOSILVER IN WISTAR RATS(Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2012-10) APARNA REDDY, N; ESWARA PRASAD, P (Major); PADMAJA, K; ADILAXMAMMA, KABSTRACT : An experimental study was conducted to evaluate and compare the wound healing properties of citrate coated silver nanoparticles and B.ovalifoliolata mediated silver nanoparticles in wistar rats in three wound models. The nanoparticles were synthesized and the synthesized nanoparticles were characterized using UV-visible spectrophotometer, Scanning electron microscope (SEM), DLS (Dynamic light scaterring) Technique and FT-IR analysis and were also evaluated for antimicrobial activity and cytotoxic effects. A total of 90 rats (both sexes) were divided into five groups, each group containing 18 rats and the wound healing activity was evaluated in three different wound models viz., excision, incision and dead space model. Group 1 was treated with normal saline, group 2 with Povidone iodine (1% solution), group 3 with Citrate coated silver nanoparticles (AgNPs) (170 μg per dressing), group 4 received B.ovalifoliolata bark ethanolic extract (50 mg per dressing) and group 5 received B.ovalifoliolata mediated silver nanoparticles (BENS) (85 μg per dressing). The respective concentrations of the drug were prepared as dressings and applied topically. In excision wound model, percentage wound contraction and the periods of epithelialization were recorded. Blood was collected from orbital sinus in EDTA coated tubes and plasma was separated for estimation of Vitamin C, TBARS and protein. Granulation tissue from excision model was processed for histopathological examination. In incision wound model, the wound breaking strength was measured on 10th day. In dead space wound model, the granulation tissue collected on Day 10 was used for the estimation of hydroxyproline, hexosamine, total protein, TBARS, superoxide dismutase, catalase, reduced glutathione and ascorbic acid. UV Visible absorption spectrum showed a maximum absorption between 350-450 nm and around 450 nm for AgNPs and BENS respectively. SEM images showed that AgNPs showed an agglomerated morphology (size 374-718 nm) while, B.ovalifoliolata mediated silver nanoparticles were spherical in shape (size 192.8-346.1 nm). The results of FT-IR analysis indicated the involvement of hydroxyl, carboxyl and amine groups in the synthesis of B.ovalifoliolata mediated silver nanoparticles. The size of the particles as measured by DLS technique was 78.8 and 273.5 nm respectively. The in vitro studies showed that BENS possess highest antibacterial activity against Staphylococcus aureus as evidenced by highest zone of inhibition (17.67 mm) and lowest MIC value (1.77μg/ml). BENS was practically nontoxic at 85 ppm while AgNPs were toxic from 10 ppm onwards. In excision wound model, BENS showed significantly (p<0.05) higher percentage of wound contraction and shortest period of epithelialization compared to control group indicating better wound healing property. The TBARS level was significantly (p<0.05) lower and the levels of vitamin C and protein were elevated in both BE and BENS groups. Histopathological examination of BENS group showed mild cellular infiltration with angiogenesis and more fibroblast proliferation when compared to control. In the incision wound model, BE and BENS groups showed significantly (p<0.05) higher wound breaking strength compared to other groups. In dead space wound model, there was a significant increase in hydroxyproline and hexosamine content with increased catalase and SOD activity and decreased lipid peroxide levels in the granulation tissue of BENS group compared to other treatment groups. Thus in conclusion B.ovalifoliolata mediated silver nanoparticles showed better wound healing properties compared to citrate coated silver nanoparticles and ethanolic extract of B.ovalifoliolata.