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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 ISOLATION OF BUFFALO URINARY PROTEINS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2007-09) RAGA SUDHA, MEDICHERLA; Ashok, VASILI(MAJOR); Viroji Rao, S.T.; Narasimha Reddy, YABSTRACT: Presence of proteins in urine is considered pathological, but still certain proteins are excreted in minute quantities into the urine of healthy animals. The normal proteins in the urine are uroplakins (15 to 47kDa) and uromodulin (85 to 90kDa) which are produced by the urinary bladder and the thick ascending limb of Henle’s loop (kidney) respectively. The tissue specific expression of these proteins in the urinary system formed the basis for transforming the urinary system into a bioreactor to produce proteins of economic importance in the urine. In the present day world, the idea of transforming urinary system as a bioreactor and urine as an expression system has growing importance. So, the objective of the present study is isolation and quantification of these normal buffalo urinary proteins. The outline of the work is collection of urine from apparently healthy lactating Murrah buffaloes. Precipitation of proteins from the urine is carried out by protein precipitating agents like acetone, ammonium sulphate and sodium chloride. These precipitated proteins were analyzed by SDS-PAGE. Coomassie blue stained gel revealed a prominent and consistent protein band of molecular weight 85kDa in all the precipitated samples which could be uromodulin, the protein of our interest. Silver nitrate stained gels revealed low molecular weight protein bands (15, 27 and 47kDa) in all precipitated samples in addition to the high molecular weight band, which could be uroplakins. The consistent production of uromodulin by the animal into the urine gives an idea of transforming the kidney into a bioreactor. The reason for inconsistent occurrence of various low molecular weight proteins could be due to the dilution effect of liquid intake by the animal, diet, temperature, climate and various physiological factors which are yet to be ruled out. Quantification of protein precipitates of urine samples and urine as such of 10 different healthy buffaloes was done. The mean protein yields (mg) of urine and precipitates of acetone, ammonium sulphate and sodium chloride are 0.96±10.20, 10.60±0.39, 1.50±0.04 and 1.91±0.13 respectively. Acetone precipitation yielded significantly (p<0.001) higher quantity of protein precipitate. However, the banding pattern is not commensurate with the amount of precipitate. This could be the result of interference of salts and metabolic wastes with the Lowry’s method, yielding higher values. Ammonium sulphate precipitated more number of proteins, evidenced by the gel replicates, but at higher concentration. Sodium chloride selectively precipitates almost a single protein and is required in small quantities when compared to ammonium sulphate for producing good banding pattern. It is concluded that acetone precipitates higher amounts of protein quantitatively. Ammonium sulphate precipitates more number of proteins, where as sodium chloride precipitation yields almost a single protein. However, sodium chloride method has the advantage of selective precipitation over acetone and ammonium sulphateThesisItem Open Access PROTEIN PROFILE OF POULTRY KIDNEY(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2007-09) KALYANI, P; ASHOK, VASILI(MAJOR); RAJASEKHAR REDDY, A; NAGAMALLESHWARI, YABSTRACT : The extensive prevalence and escalating incidence of Chronic Renal Failure and End Stage Renal Disease worldwide necessitates renal tissue engineering as a practical solution to meet the organ demand for renal transplantation therapy. Renal tissue engineering is a multidisciplinary field, which is supported on three pillars- cells, scaffolds and signaling biomolecules. A critical understanding of the role of signaling biomolecules is necessary for tissue engineering because these biomolecules are differentially expressed with specific spatio-temporal distribution. Signalling biomolecules and signal transduction pathways are highly conserved between species during millions of years of evolution, which engenders the scope for probing into mammalian kidney biology by using lower organisms as models. Gallus gallus (chicken) is a suitable animal model to study the role of proteins in renal organogenesis, owing to the availability of extensive literature on embryogenesis and the ease of embryo retrieval. Metanephric kidneys were collected from different developmental stages of Vanaraja breed chick embryos viz., on 7th , 9th , 11th , 13th , 15th , 17th , 19th days , day old chicks and adult cock in ice cold PBS. Embryonic kidneys and kidneys of day old chicks were dissected under diascopic stereo zoom microscope. The kidney tissues were homogenized, centrifuged and supernatants were analyzed by SDS PAGE. Prominent, differential protein expression was evident upon comparing the protein bands of tissue extracts. In total, 22 prominent differentially expressed proteins were identified on careful examination of banding pattern from the tissue extracts of different developmental stages. A single high molecular weight protein band was seen only in the adult sample. Some of these prominent protein bands were identified to be MMP9 (97 kDa), nephronectin (79 kDa), osteopontin (76 kDa), MMP2 (72 kDa), pax 8 (57 kDa), pax 2 (50 kDa), BMP 4 (45 kDa), actin (43 kDa), FGF2 (23kDa), pleitrophin (21kDa), activin 1 (20kDa), endostatin (15kDa) based on their molecular weights and temporal distribution. Further confirmation of the identity of these proteins could be done by in vitro cultural tests, protein sequencing and other functional tests. The present study reveals a prominent, differential protein expression at various developmental stages in kidney organogenesis. Based on the banding pattern, it is proposed that most of the architectural design for kidney organogenesis is framed by the end of embryonic day 15 and after that it is refined to perfection to carry out various biological activities. Research on the histology and immuno-histochemistry of the kidney tissue during various developmental stages can confirm the present findings.ThesisItem Open Access EFFICACY OF ACTIVATED CHARCOAL AND YEAST CULTURE ON AMELIORATION OF COMBINED TOXICITY OF AFLATOXIN AND T-2 TOXIN IN BROILERS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2007-01) RAGHAVENDRA SRIKANTH, N; NARASA REDDY, G.V(MAJOR); SETTY, O.H.; RAJASHEKAR REDDY, AABSTRACT : Aflatoxin and T-2 toxin were produced from Aspergillus parasiticus and Fusarium sporotrichioides (F. tricinctum) respectively and quantified using TLC. The toxins were mixed with broiler feed to attain required concentrations. Four diets for broilers were prepared diet 1 – basal diet (control), diet 2-basal diet + aflatoxin (1 ppm) + T-2 toxin (1 ppm), diet 3 – basal diet + aflatoxin (1 ppm) + T-2 toxin (1 ppm) + activated charcoal (0.4%), diet 4 – basal diet + aflatoxin (1 ppm) + T-2 toxin (1 ppm) + activated charcoal (0.4%) + yeast culture (0.1%). These four diets were fed to 4 groups of day old broiler chicks with four replicates of eight birds in each replicate, in a completely randomized design for six weeks. The body weight gains were significantly (P<0.01) lower on diet 2 containing aflatoxin and T-2 toxin. The body weight gains increased gradually on diet 3 containing activated charcoal and on diet 4 containing activated charcoal and yeast culture. Feed consumption was significantly (P<0.01) higher on basal diet compared to all other diets, which were at par. Feed conversion ratio was the highest on diet 2 compared to other diets which were at par. On diet 2 containing aflatoxin and T-2 toxin, the biochemical profile revealed elevated serum enzyme activities of AST, ALT, ALP, GGT indicating liver toxicity. These studies also recorded decreased levels of serum proteins, glucose, cholesterol, triglycerides, calcium, uric acid and increased levels of serum bilirubin. Increased serum creatinine and BUN recorded indicates toxicity to kidney. The studies on liver and kidney function tests indicate possible damage of these organs in the chicks fed on diet 2. Degenerative changes in liver, kidney and lymphoid depletion in bursa of Fabricius, disruption of cardiac muscle fibres, and disruption of intestinal villi were observed on histopathology in birds fed with aflatoxin and T-2 toxin supporting the biochemical study. The birds on diet 3 showed lower body weight gains and feed consumption compared to that of control diet. Efficiency of feed utilization on diet 3 was comparable to that of diet 1. The biochemical parameters like AST, ALP, GGT, serum calcium, serum uric acid were moderately improved but there was no improvement in creatinine and bilirubin compared to diet 2. There was mild improvement in serum proteins cholesterol, glucose and triglyceride levels compared to diet 2. Complete alleviation of toxic symptoms was noticed in terms of serum ALT and BUN. Broilers on diet 4 showed an increase in body weight gains compared to diet 2. Feed conversion ratio on diet 4 was comparable to that on diet 1. The biochemical profile of cholesterol total protein, glucose, uric acid, calcium and triglyceride showed moderate improvement while the other parameters like ALP, bilirubin, ALT, GGT and AST were comparable to those of control diet. Histopathological studies were comparable to those of control group fed on toxin free diet. These results indicate that aflatoxin and T-2 toxin in the diet caused deteriorated effects on growth, biochemical profile and caused damage to vital organs. The combination of activated charcoal and yeast culture was more effective in counteracting the combined toxicity of aflatoxin and T-2 toxin compared to the activated charcoal alone.ThesisItem Open Access DETOXIFICATION OF CITRININ AND FUMONISIN IN FEED AND ITS EFFECT ON SERUM BIOCHEMICAL PROFILE IN BROILERS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2007-01) ASHOK KUMAR, DEVARASETTI; ASHOK, VASILI(MAJOR); NARASA REDDY, G.V; ANJANEYULU, YABSTRACT : Citrinin and fumonisin toxins were produced from Pencillium citrinum and Fusarium moniliforme respectively and both were quantified using Thin Layer Chromatography (TLC) method. The toxins citrinin @ 50 ppm and fumonisin @ 10 ppm were mixed in broiler feed to attain required concentrations. Four diets for broilers were prepared diet 1 – basal diet (control), diet 2 – basal diet + citrinin (50 ppm) + fumonisin (10 ppm), diet 3 – basal diet + citrinin (50 ppm) + fumonisin (10 ppm) + activated charcoal (0.4%) and diet 4 – basal diet + citrinin (50 ppm) + fumonisin (10 ppm) + activated charcoal (0.4%) + lyophilized yeast culture (0.1%). Each diet was fed to a group of day old broiler chicks with four replicates having eight birds in each replicate in a completely randomized design for six weeks. The weekly body weights and weekly body weight gains were significantly (P < 0.01) lower on diet 2 containing citrinin (50 ppm) + fumonisin (10 ppm). The weekly body weights and weekly body weight gains increased gradually on diet 3 containing activated charcoal. Further significant (P < 0.01) improvement was recorded on diet 4 containing activated charcoal and yeast culture. Feed consumption was significantly higher (P < 0.01) on basal diet compared to other test diets. Feed conversion ratio was the highest on diet 2 when compared to other test diets. On diet 2 containing citrinin and fumonisin, the biochemical profile revealed elevated serum enzyme activities of AST, ALT, ALP, GGT indicating liver toxicity. These studies also recorded decreased levels of serum proteins, albumin, glucose, cholesterol, triglycerides, calcium, uric acid and increased levels of serum bilirubin. Increased serum creatinine and BUN recorded indicates toxicity to kidney. These studies on liver and kidney function tests indicate possible damage of these organs in the chicks fed on diet 2. Further, supporting the biochemical study, degenerative changes and central venous congestion in liver and marked degenerative changes in kidney and lymphoid depletion in bursa of Fabricius, disruption of cardiac muscle fibres, disruption of intestinal villi were observed on histopathology in birds fed with citrinin and fumonisin. The birds on diet 3 containing citrinin, fumonisin and activated charcoal showed significant (P < 0.01) improvement in body weight gains and feed consumption. Efficiency of feed utilization on diet 3 was comparable to that of diet 1. The serum enzymes like AST, ALT, ALP and GGT were markedly improved and serum creatinine, BUN, calcium, serum uric acid were moderately improved when compared to diet 2. There was mild improvement in serum proteins, cholesterol, glucose and triglyceride levels compared to diet 2. All other biochemical parameters and histopathological findings showed improvement compared to toxin fed group (diet 2). This indicates that activated charcoal (0.4 %) had partial amelioration on toxic effects of citrinin and fumonisin. The birds fed on diet 4 recorded the serum enzymes like AST, ALT, ALP and GGT in the liver nearer to the control group (diet 1). All the biochemical parameters and histopathological findings were also nearer to that of the control group and within the normal range indicating that activated charcoal and lyophilized yeast culture had a complementary effect in ameliorating the toxic effect of citrinin and fumonisin. These results indicate that activated charcoal (0.4 %) could adsorb and ameliorate the toxins (citrinin and fumonisin) to some extent and activated charcoal (0.4 %) and lyophilized yeast culture (0.1 %) showed complementary effect in amelioration of the toxic effect caused by citrinin and fumonisinThesisItem 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 urine