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Anand Agricultural University, Anand
Anand Agricultural University (AAU) was established in 2004 at Anand with the support of the Government of Gujarat, Act No.(Guj 5 of 2004) dated April 29, 2004. Caved out of the erstwhile Gujarat Agricultural University (GAU), the dream institution of Sardar Vallabhbhai Patel and Dr. K. M. Munshi, the AAU was set up to provide support to the farming community in three facets namely education, research and extension activities in Agriculture, Horticulture Engineering, product Processing and Home Science. At present there seven Colleges, seventeen Research Centers and six Extension Education Institute working in nine districts of Gujarat namely Ahmedabad, Anand, Dahod, Kheda, Panchmahal, Vadodara, Mahisagar, Botad and Chhotaudepur
AAU's activities have expanded to span newer commodity sectors such as soil health card, bio-diesel, medicinal plants apart from the mandatory ones like rice, maize, tobacco, vegetable crops, fruit crops, forage crops, animal breeding, nutrition and dairy products etc. the core of AAU's operating philosophy however, continues to create the partnership between the rural people and committed academic as the basic for sustainable rural development. In pursuing its various programmes AAU's overall mission is to promote sustainable growth and economic independence in rural society. AAU aims to do this through education, research and extension education. Thus, AAU works towards the empowerment of the farmers.
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ThesisItem Open Access SUBACUTE TOXICOPATHOLOGICAL STUDIES OF TAMOXIFEN IN WISTAR RATS(AAU, Anand, 2011) PANCHAL, VIJAY P.; Ghodasara, D. J.The present research work was conducted on 24 male and 24 female Wistar rats to study the toxicoathological effects of repeated dose (28 days) of tamoxifen. Wistar rats were randomly divided into 4 different groups with six males and six females in each group. Animals of group II to IV were given 1, 10 and 20 mg/kg b.wt tamoxifen by oral gavage for 28 days where as group I was administrated only 0.5% CMC as (vehicle) control. After completion of 28 days treatment, blood samples were collected for haematology and serum biochemical analysis from retro-orbital plexus with the help of capillary tube. The animals were sacrificed by high dose of anesthesia with Di - ethyl ether on 29th day for necropsy and collection of tissue. Necropsy examination was performed in all sacrificed animals and gross lesions were recorded. Tissue samples (lung, liver, kidney, intestine, spleen, testes, epididymis, heart, brain and uterus) were collected in 10% formalin solution for histopathological examination. The extent and severity of observed symptoms varied according to the dosage administered to animals. Symptoms like weakness, loss of appetite, aggressiveness and mild alopecia were noticed in rats of high dose group. The dose dependent reduction in body weight and feed consumption were observed in animals of group II, III and IV. The significant decrease in RBC count, packed cell volume, haemoglobin and MCV was recorded in group IV whereas significant increase in total leucocyte count was noticed in group III and highly significant increase in group IV animals. The differential leucocyte count revealed significant increase in neutrophil count in group III and highly significant increase in group rV animals whereas significant decrease in lymphocyte count in animals of tamoxifen treated group IV. No significant change in monocyte, eosinophil and basophil counts were observed in tamoxifen treated groups. AST and ALT values increased significantly in group III and highly significantly in group IV. The significant increase in AKP, creatinine and BUN values were recorded in treatment group IV. The significant decrease in total protein and albumin were observed in treatment group HI and highly significant decrease in group IV. All the rats exposed to tamoxifen at three different dose levels revealed dose dependant pathological changes in group III and IV in different organs. The lesions were characterized by degeneration, necrosis, inflammatory and vascular changes. The main target organs affected were liver, testes and uterus. The overall lesions gave impression that tamoxifen was hepatotoxic as well as toxic to reproductive system. The intensity and distribution of such lesions were more severe in rats of group TV followed by group III.ThesisItem Open Access PHENOTYPIC AND GENOTYPIC IDENTIFICATION AND METAGENOMIC ANALYSIS OF SUBCLINICAL MASTITIC PATHOGENS IN COWS(AAU, Anand, 2011) BHANDERI, BHARAT BABUBHAI; Jhala, M. K.Subclinical mastitis occurs with no visible changes in the appearance of the milk and/or the udder, but milk production decreases which leads to economic losses to the farmers and dairy industry. There are many microbial pathogens involved in causing subclinical mastitis in cows. The present study was undertaken to know incidences of subclinical mastitis in organized farms using Somatic Cell Count (SCC) and bacteriological examination (International Dairy Federation-IDF guidelines), California Mastitis Test (CMT) and impregnated pH strip test followed by characterization and PCR based detection of important mastitic pathogens. Metagenomic analysis of subclinical mastitis milk was also done to determine the complex microbial diversity in udder environment during subclinical mastitis. A total of 349 quarters of 89 lactating cows comprising 31 Triple cross (TP) (Kankrej x Jersey x Holstein Friesian), 29 Kankrej, 17 Gir and 12 Holstein Friesian (HF) affiliated with Anand Agricultural University, Anand were screened for subclinical mastitis. Overall 52.8 per cent (47/89) cows were found to be positive for subclinical mastitis infection in one or more quarters. The highest incidence of subclinical mastitis was found in Triple cross cows (74.19%), followed by Gir cows (58.82%), HF cows (50%) and Kankrej cows (27.58%). Overall quarter wise incidence for subclinical mastitis was found to be 30.66 per cent (107/349). The highest incidence was found in Gir cows (38.80%) followed by Triple cross cows (38.08), HF cows (33.33%) and Kankrej cows (15.04%). The highest incidence of subclinical mastitis was found in fore left quarter (28.03%), followed by hind left quarter (27.1%), fore right quarter (24.29%) and hind right quarter (20.56%). Of the 47/107 cows/quarters positive for subclinical mastitis, 39/47 (82.97%) cows and 82/107 (76.63%) quarters were also positive by CMT and 27/47 (57.44%) cows and 56/107 (52.33%) quarters were positive by impregnated pH strip test. Cultural isolation ft'om 107 subclinically positive quarter milk samples yielded 126 bacterial isolates. Staphylococci was the most predominant bacterial species accounting for 53.97 per cent (68/126) of all the isolates, followed by 21.43 per cent (27/126) CAMP (Christie-Atkins-Munch-Peterson) test positive Str. agalactiae, 18.25 per cent (23/126) Micrococci, 4.77 per cent (6/126) E. coli and 1.58 per cent (2/126) Bacillus species. Out of 68 Staphylococci isolates, 38 (55.89%) isolates showed fermentation on Mannitol Salt Agar (MSA), whereas 30 (44.11%) isolates were mannitol non fermentive. Of the total 30 S. aureus identified by PCR, 21 (70%) were mannitol fermentive and 9 (30%) mannitol non fermentive. Thirty one (45.58%)) Staphylococci were found to be positive for pigment production, whereas 37 (54.42%) isolates produced white colonies on nutrient agar. Forty eight (70.58%) isolates were found positive for coagulase reaction, whereas 20 (29.41%) were negative. Thirty one (45.58%)) isolates exhibited P haemolysin production, 4 (5.89%) a haemolysin and 33 (48.53%)) isolates were non-haemolytic on 5 per cent Sheep blood agar. Phage typing at National Staphylococci Phage typing Centre, Maulana Azad Medical College, New Delhi, using five phage group sets of International Basic Set of 23 phages revealed maximum number of the Staphylococci isolates lysed by group II 14 (82.35%), followed by groups III, Not alloted (NA), I and V with 12 (70.58%), 9 (52.94%), 5 (29.23%) and, 2 (11.76%) respectively. Maximum 11 (64.7%) isolates were lysed with phage number 47 with strong reaction, followed by 10 (58.82%)) isolates with phage numbers 42E and 81, while less effective phage numbers were 71 and 94, which lysed only one strain (5.89% each) and phage number 95 not giving strong reaction with any of the isolates. The methicillin and oxacillin antibiotic sensitivity pattern by disc diffusion method revealed that, all the 68 (100%)) Staphylococci isolates were sensitive. Serotyping of six E. coli isolates (at National Salmonella and Escherichia Centre, Kasauli, Himachal Pradesh for 'O' antigen) resulted in identifying 014, O20, 045, 055 and 0112 serotypes, while one isolate was untypeable (UT). Out of 68 Staphylococci isolates tested for identification of 5. aureus by PCR, 30 isolates were identified as S. aureus by obtaining amplification product of 1318bp using S. aureus specific primer for 23S rRNA. Out of 30 PCR positive S. aureus, 18 (60%)) were positive and rest were negative for coagulase test. All the 27 Streptococci isolates were identified as Str. agalactiae by amplifying 586bp product using Str. agalactiae specific primer for the 16S rRNA while, none were amplified for Str. dysgalactiae (401bp) and Str. uteris (94bp) based on primers specific for the 16S rRNA and 23 S rRNA respectively. All the six E. coli isolates yielded 232bp amplified product using E. coli specific primer targeting DNA sequence coding for the 23 S rRNA. Metagenomic analysis (using GS FLX 454 Life Sciences) of DNA of subclinical mastitis milk sample of TP, Kankrej and Gir cows yielded an out put of 274190 bp, 17,727 bp, 42,548 bp and 1,960, 170, 301 contigs respectively. Average fragment length obtained were 139.89, 104.28 and 141.36 bp for TP, Kankrej and Gir cows respectively. The longest sequence length was 560, 327 and 454 bp, while shortest sequence length was 40, 40, and 41 bp for TP, Kankrej and Gir cows respectively. A total of 54 (2.76%), 39 (22.94%) and 12 (3.99%) sequences for TP, Kankrej and Gir cows respectively could be matched to proteins in SEED subsystems of MG-RAST (Meta Genome Rapid Annotation with Subsystem Technology) (using an e-value cut-off of le-5). Metagenomic analysis of the three breeds identified bacterial organisms belonging to phyla (5), class (8), Subclass / order (15), Family (19), Genus (23) and species (28); of these, 19 genera and 26 species, many of which were fastidious/anaerobic organisms, were identified additionally than the cultural methods. Out of five genera Staphylococcus, Streptococcus, Micrococcus, Bacillus and Escherichia detected in the subclinical mastitis milk samples of TP, Gir and Kankrej breeds by culture based methods, four genera Staphylococcus, Streptococcus, Bacillus and Escherichia were also identified in the corresponding pyrosequencing data, while Micrococcus identified by culture based methods was not found in the pyrosequencing data. In pyrosequencing, over all 28 bacterial species were identified from all the three breeds of cows viz. Leifsonia xyli, Propionibacterium acnes, Streptomyces coelicolor, Chlamydophila abortus, Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus acidophilus, Streptococcus mitis, Burkholderia cenocepacia, Burkholderia cepacia, Ralstonia solanacearum, Nitrosomonas europaea, Pseudoalteromonas atlantica. Salmonella Dublin, Serratia marcescens, Azotobacter vinelandii, Pseudomonas aeruginosa, Pseudomonas mendocina, Stenotrophomonas maltophilia. Bacillus subtilis, Lactobacillus delbrueckii. Aster yellows witches'-broom phytoplasma, Pannbaculum lavamentivorans, Thermosipho melanesiensis, Aeromonas hydrophila, Escherichia coli, Shigella hoydii and Pseudomonas fluorescens. Of these, except S. aureus and E. coli, all were additionally identified than the culture based method but, Str. agalactiae identified by cultural method was not found in the pyrosequencing data. The role of lesser known or less frequently involved organisms as identified by metagenomic analysis may be further explored in future so as to understand the complete etiopathology of subclinical mastitis in cows.ThesisItem Open Access TOLL LIKE RECEPTORS (TLR) EXPRESSION IN MILK SOMATIC CELLS DURING MASTITIS IN CATTLE USING REAL-TIME PCR(AAU, Anand, 2011) GHADIGAONKAR, DINESH DILIP; Rank, D. N.The dairy sector in India has shown remarkable progress in the recent years and consequently the country has now become the largest milk producer and valueadded milk products in the world. Though the country is the largest producer of milk, milk production per animal is much less compared to agriculturally developed countries. One of the reasons for this less milk production per animal is loss of milk production capacity because of inflammation of the mammary gland or mastitis in dairy animals. Mastitis is defined as an inflammatory reaction of the parenchyma of the mammary glands to bacterial, chemical, thermal or mechanical injury which is characterized by a range of physical, chemical and usually bacteriological changes in the milk and pathological changes in the glandular tissue which is the most common and the most expensive disease of dairy cattle throughout the world. Mastitis is classified as subclinical and clinical mastitis. The cows that have subclinical mastitis are reservoirs of organisms that lead to infection to other cows. Most clinical cases start as subclinical; thus, controlling subclinical mastitis is the best way to reduce the clinical cases. Somatic cells consist of leukocytes and epithelial cells exfoliated from the mammary epithelium. Mastitis is associated with an influx of inflammatory cells; hence somatic cell count of milk increases. Enumeration of somatic cell counts and bacterial culture of milk has been suggested as a standard method for detecting subclinical udder infections in dairy cows. Genes have major impact on health status of animals. Genetic variability of mastitis resistance is well established in dairy cattle. Resistance to mastitis is a complex function involving various biological pathways, molecules and cells. Study of the expression of genes involved in mastitis resistance is major tool for early diagnosis of disease and genetic improvement to superior stock. Toll Like Receptors (TLR) are cell-surface receptors that recognizes a broad class of pathogen-associated molecular patterns, activates immune system, and induces the over expression of inflammatory factors, which participate in irmate immune responses to confer disease resistance. The bovine TLR genes have been studied in recent years. Hence the study was planned with the objective to investigate expression of TLR-2, TLR-4, TLR-9 in somatic cells in healthy and mastitic udder by Real Time PCR in cattle. The study was undertaken to assess different TLRs (TLR-2, TLR-4 and TLR- 9) in three cattle breeds namely Gir, Kankrej and Triple crossbred (Kankrej x Jersey x Holstein Friesian) with sub-clinical mastitis. A total of 65 lactating cows comprising 16 Gir, 29 Kankrej and 20 Triple crossbred animals were screened for presence of mastitis using Electronic Somatic Cell Counter and bacteriological culture examination. Total RNA was extracted from milk somatic cells from 15 positive and 6 healthy quarters from each breed using TRIZOL method. The RNA was treated with DNase enzyme to remove any traces of genomic DNA. cDNA was synthesized from RNA using Qiagen's Omniscript reverse transcriptase kit and random hexamer primers. The amplification of cDNA template of TLR 2, TLR 4 and TLR 9 genes was carried out using gene specific primers. Expression of TLR 2, TLR 4 and TLR 9 genes mRNA was quantified by Real Time PCR and analysed using Applied Biosystems 7500 SDS software. Relative expression study of these genes was carried out using GAPDH as internal control. Results indicated that there was upregulation of TLR 2, TLR4 and TLR9 gene expression in animals affected with subclinical mastitis compared to healthy animals. Targeted amplification of 421 bp TLR 2, 108 bp TLR 4 and 108 bp TLR 9 was confirmed by agarose gel electrophoresis. Prevalence of subclinical IMI was higher in Triple crosbred cows (65%) compared to Gir (50%) and Kankrej cows (27.59 %). The mean SCC of infected quarters was significantly higher than that of noninfected quarters (P < 0.05) in all three breeds. The average relative expression of all three genes i.e. TLR2, TLR4 and TLR9 was higher (ranged from 7 to 35 folds) in mammary gland with subclinical intramammary infection than those measured in the uninfected glands. The concomitant increase in somatic cell count and upregulation of TLR2, TLR4 and TLR9 gene expression was observed during subclinical mastitis in all three breeds. Comparison of SCC upregulation between breeds indicated that, there was no significant difference between breeds in the SCC in the diseased quarter during subclnical mastitis in Gir, Kankrej and triple crossbred cows. In Gir cows, TLR2 gene expressions level in diseased quarters was found to be upregulated with an average 10.54 (10.54 ± 7.12) fold compared to pooled healthy quarters. In Kankrej cows, TLR 2 gene expressions level in diseased quarters was found to be upregulated with an average 12.22 (12.22 ± 11.61) folds compared to pooled healthy quarters. In Triple crossbred cows, TLR 2 gene expression level in diseased quarters was found to be upregulated with an average 7.13 (7.13 ± 10.57) folds compared to pooled healthy quarters. In Gir cows, TLR 4 gene expressions level in all diseased quarters was found to be upregulated with an average 18.43 (18.43 ± 24.230) fold upregulation compared to pooled healthy quarters. In Kankrej animals, TLR 4 gene expressions level in diseased quarters was found to be upregulated with an average 31.59 (31.59 ± 18.74) folds compared to pooled healthy quarters. In Triple crossbred cows, TLR 4 gene expression level in diseased quarters was found to be upregulated with an average 23.817 (23.817 ± 27.6963) fold compared to pooled healthy quarters. In Gir cows, TLR 9 gene expression level in diseased quarters was found to be upregulated with an average 6.193 (6.193 ± 8.19) fold compared to healthy quarters. In Kankrej cows, TLR 9 gene expression level in diseased quarters was found to be upregulated with an average 5.44 (5.44 ± 8.14) folds compared to Pooled healthy quarters. In Triple crossbred cows, in all diseased quarters, TLR 9 gene expression level was found to be upregulated with an average 19.29 (19.29 ± 16.31) fold compared to pooled healthy quarters. During subclinical mastitis SCC was found to be positively correlated with the transcriptional activities of TLR2, TLR4 and TLR9 gene in Gir and Triple crossbred cow. In Kankrej cows TLR 2 and TLR 9 gene expressions were positively correlated with s e c but TLR 4 gene expression was not correlated with SCC. The level of infection as reflected by number of somatic cells had significant effect on level of upregulation in gene expression. However, there was no significant effect of a breed on level of upregulation of TLR 2, TLR 4 and TLR 9 gene expression.ThesisItem Open Access ISOLATION, IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF BACTERIAL PATHOGENS FROM RESPIRATORY TRACT OF APPARENTLY HEALTHY AS WELL AS SICK GOATS(AAU, Anand, 2011) AHER, TUSHAR KISAN; ROY, ASHISHA number of factors are responsible for economic losses to the goat industry; among them the pneumonia due to various bacterial species imposes serious constraints on goat production all over the world because of high mortalities. The major health problem of small ruminants is pneumonia/pleuropneumonia, which may be caused by Mycoplasma and Pasteurella species alone or in conjunction with other microbes. Pneumonia in small ruminants constitutes a serious setback to the growth in this group of animals with resultant economic losses in many parts of the world. Thus, the present study was undertaken with a view to know preponderance of this bacterial spp. in relation to respiratory tract infections in apparently healthy and sick goats. The objectives were isolation, identification, nucleic acid based detection of virulence associated and toxigenic potentials and in vitro antibiotic sensitivity patterns of the isolates from respiratory tract infections of apparently healthy as well as sick goats. In the present investigation, total 102 nasal swab samples and 96 tissue samples were collected from apparently healthy as well as sick goats. Bacterial isolation was done following standard technique by inoculating tissue sample and nasal swab sample primarily on blood agar and plates incubated for 24-48 hrs at 37°C. After incubation, the nature of growth and cultural characters of colonies were studied. Preliminary morphological identification was based on Gram's staining. Specific identification and biochemical characterization of the isolates was done as per the standard techniques. In this study, ten different types of bacteria were isolated. It includes Mycoplasma spp. (0.7%), P. inultocida (0.7%), Staphylococcus spp. (29.9%), Micrococcus spp. (4.2%), Streptococcus spp. (9.7%), Bacillus spp. (19.4%), E. coli (18.8%), Proteus spp. (4.9%), Klebsiella spp. (5.6%) and P. aeruginosa (6.3%). The most prevalent species of bacteria found was Staphylococcus spp. Gram positive organisms were more prevalent in apparently healthy goats (46.5%) than sick goats (11.8%). Gram negative organisms were more prevalent in sick goats (24.3%) than apparently healthy goats (16.7%). From 102 nasal swab samples- 68 isolates, 32 lung samples- 31 isolates, 32 trachea samples- 26 isolates, 32 tonsil samples- 19 isolates were obtained. Out of which. Gram positive bacteria were 91 (63.2%), whereas Gram negative bacteria was 52 (36.1%) and a single isolate was identified as Mycoplasma spp. (0.7%). From 102 nasal swabs, total 68 isolates were obtained and there were total nine different types of bacteria isolated, viz.. Mycoplasma spp. (1.5%), P. multocida (1.5%), Staphylococcus spp. (38.2%). Micrococcus spp. (8.82%), Streptococcus spp. (7.4%), Bacillus spp. (33.8%), E. coli (5.9%), Klebsiella spp. (1.5%) and P. aeruginosa (1.5%). The most pre\alent bacterial species found in nasal swab were Staphylococcus spp. From. 32 lung samples, total 31 isolates were obtained and there were total seven different types of bacteria isolated, viz., Staphylococcus spp. (19.4 %). Streptococcus spp. (12.9%), Bacillus spp. (6.5%), E. coli (35.5%), Proteus spp. (6.5%), Klebsiella spp., (9.7%) and P. aeruginosa (9.7%). The most prevalent bacterial species found in lung was E. coli. From 32 tracheal samples, total 26 isolates were obtained and there are total six different types of bacterial species were isolated. It involves Staphylococcus spp. (30.8 %), Streptococcus spp. (7.7%)), Bacillus spp. (11.5%), E. coli (34.6%), Proteus spp. {1.1%) and P. aeruginosa (1.1%). The most prevalent bacterial species found in trachea was E. coli. From 32 tonsillar samples, total 19 isolates were obtained and there are total six different types of bacterial species were isolated. It involves Staphylococcus spp. (15.8 %), Streptococcus spp. (15.8%), E. coli (15.8%o), Proteus spp. (15.8%), Klebsiella spp., (21.1%) and P. aeruginosa (15.8%)). The most prevalent bacterial species found in tonsil was Klebsiella spp. Molecular characterization of the isolates by PCR based method was applied for specific detection as well as detection of virulence associated and toxigenic genes.ThesisItem Open Access EFFECT OF ENZYME SUPPLEMENTATION IN LAYER DIETS ON EGG PRODUCTION PERFORMANCE OF TWO STRAINS OF WHITE LEGHORN CHICKEN(AAU, Anand, 2011) Patel, Atulkumar Babubhai; Khanna, KuldeepExperimental population included "FWN" and "IWP" strains of White Leghorn type laying hens of seventh generation. 360 pedigreed females of IWN strain and 360 pedigreed females of IWP strain were used for the experiment. Various traits required for present study were measured at different periods of age. As dietary energy level was increased, body weight (BW) was increased significantly (P < 0.05) at 40, 64 and 72 weeks of age. Energy levels (2400, 2550 and 2700 kcal ME) did not affect total egg number produced per bird (TEN) significantly. As dietary energy level was increased, total feed consumption per bird (TFC) and feed consumed per egg produced (FCE) was decreased significantly (P< 0.05) at 40, 64 and 72 weeks of age. Values of feed cost per egg produced (ECOST) differed significantly (P< 0.05) at 2400, 2550 and 2700 kcal ME levels. Different energy levels did not affect egg weight [EW] at 40 [EW40] and 64 [EW64] weeks of age significantly. EW72 at 2400 kcal ME level differed significantly (P< 0.05) from EW72 at 2550 and 2700 kcal ME levels. Enzyme supplementation in layer diet lowered the BW in enzyme group than control group which was non significant. TEN was increased Avlth enzyme supplementation in layer diet but it was non significant. Enzyme supplementation had significantly (P< 0.05) reduced TFC64, TFC72, FCE64 and ECOST64. There was non significant reduction in EW in enzyme group. Effects of energy levels and enzyme supplementation were found non significant for BW. Effects of same were found significant (P< 0.05) for TEN64 and TEN72 at 2550 kcal ME level, whereas effects of same were found non significant for TEN40. Effects of energy levels and enzyme supplementation were found significant (P< 0.05) for TFC40 at 2400 and 2550 kcal ME levels but it was non significant for TFC64 and TFC72. At 2400, 2550 and 2700 kcal ME levels; TFC64 and TFC72 were lower in enzyme group than control group. Effects of same were found significant (P< 0.05) for FCE64, ECOST64 and ECOST72 at 2550 kcal ME level. Effects of same were found non significant for EW. Effects of strain of layer birds and energy levels were found non significant for BW, TEN, TFC, FCE, ECOST and EW. Effects of strain of layer birds and enzyme supplementation were found non significant for BW, TEN, TFC and EW. Effects of same were found significant (P< 0.05) for FCE64 and ECOST64 in IWP strain, whereas effects were non significant in IWN strain. Effects of strain of layer birds, energy levels and enzyme supplementation were found non significant for BW, TEN, TFC, FCE, ECOST and EW. Birds fed 2400 kcal ME/kg diet along with enzyme supplementation gave higher values of retention coefficient for dry matter, crude protein, organic matter and calcium in comparison to the same diet when fed without enzyme supplementation. Results observed from present study are in favour of enzyme supplementation in layer diet for better production performance in IWN and IWP strains. Results obtained also indicate that economical rearing of layer birds can be done with low energy (2400 kcal ME/kg) layer diets without significantly affecting production performance of IWN and IWP strains as compared with 2550 and 2700 kcal ME/kg layer diets. Enzyme supplementation can also be useful for better nutrient utilization by layer birds of IWN find IWP strains with low energy (2400 kcal ME/kg) layer diets.ThesisItem Open Access Study on Effect of Feeding Bypass Protein based Total Mixed Ration on Performance of Growing Crossbred Calves(AAU, Anand, 2011) Arewad, Gajanan Ramrao; Pandya, P. R.Sixteen crossbred (Holstein Friesian x Kankrej, Jersey x Kankrej and Holstein Friesian x Jersey x Kankrej) calves were selected from the farms of Livestock Research Station, Anand Agricultural University, Anand and were individually fed for 15 days preliminary feeding and 120 days experimental period to meet their energy and protein requirement as per NRG (2001) standards. They were randomly divided into two groups and assigned to two dietary treatments (T1 and T2) on age and body weight basis and were fed compound concentrate mixture based total mixed ration formulated as per BIS Type II standard. Total mixed ration contained compound concentrate mixture and mature pasture hay (Dicanthium annulatum) in the ratio of 50:50. The calves under the control group (TO were given calculated quantity of total mixed ration having concentrate mixture without bypass protein source, whereas, the calves under treatment group (T2) were given calculated quantity of totalmixed ration having concentrate mixture with formaldehyde treated bypass protein source. The initial body weight was 104.50 ± 1.23 and 103.46 ± 2.01 kg in T1 and T2 groups, respectively. The average total gain in body weight was 50.04 ± 0.09 and 61.13 ± 0.17 kg in T1 and T2 groups during the entire experiment, while the respective average daily gain was 421.00 ± 0.01 and 514 ± 0.02 g indicating the daily weight gain was statistically (P<0.05) significant in T2. However, the gain in heart girth of T2 calves was similar with T1 group; Also, the groups did not differ with respect to gain in body length and height.ThesisItem Open Access CLINICAL STUDIES ON ETIOPATHOLOGY AND MEDICO SURGICAL MANAGEMENT OF RECURRENT GENITAL PROLAPSE IN COWS(AAU, Anand, 2011) BADGUJAR, CHANDRAVADAN LAXMAN; TANK, P. H.Incidences of pertparturient cervico-vaginal prolapse (CVP) have been recorded in dairy cows all over the world by many workers. However, rarely there has been a mention of cases of chronic prolapse occurring beyond three months of parturition. On the contrary, it was experienced to be occurring in high number in the Gaushalas and Panjarapoles of Gujarat. A majority of the affected cows were from Gir or Kankrej breeds and their crosses. They had suffered repeated episodes of the problem. Conventional methods of treatment were unsuccessful to address this problem. Hence, a clinical study was undertaken to evaluate various aspects of CVP including medicosurgical management by different approaches in 46 cows of Gaushalas and Panjarapoles in Saurashtra region of Gujarat. A novel method of 100 point score for each character was evolved to assess the health of cow and severity of prolapse. The cows that fell in the range of aggregate points between 0-25, were graded as Grade-I; between 26-50, graded as Grade-II; between 51-75, graded as Grade-Ill, and cows with score from 75-100, were graded as Grade-IV. None of the cases under study fall in the Grade I category. The cases of Grade-II were subjected to surgical treatment utilizing Technique No. 1 (Fixation to prepubic tendon) or No. 2 (Button suture fixation). Cases falling under the Grade-Ill were treated with the use of surgical Technique No. 3 (Amputation of prolapsed mass) or No. 4 (Submucosal resection of vagina). In cases of Grade-Ill, the cows that had irreducible adhesions of the prolapsed mass or abnormalities of os cervix in the form of kinked, hardened, fibrosed or extensive enlarged cervix were subjected to Technique No. 3 (Amputation of prolapsed mass). Cases of Grade-III that had less changes of the os cervix as mentioned but had more of vaginal proliferation and fibrosis were subjected to Technique No. 4 (Submucosal resection of vagina). The cases of Grade-IV were put to treatment by Technique No. 5 (Pervaginum Panhysterectomy). These cases were advanced, unmanageable, highly suffering and with non-viable appearance of prolapse. Those cows which showed ovarian abnormalities were subjected to Technique No. 6 (Pervaginum Bilateral ovariectomy). They came in Grade II prolapse. After supportive therapy, anaesthesia was performed in the form of caudal epidural analgesia and pudendal nerve block under xylazine sedation. Appropriate surgical treatment was applied to each cow. The 46 affected cows were grouped as per the surgical technique provided from Group 1 to VI. In Group I to V, eight cases were operated, while in Group VI, six cases were operated. In addition to this, blood and serum samples were collected for comparison, from eight healthy lactating normal cows as a control group. For fixation of vagina to prepubic tendon, insertion of the needle in the prepubic tendon required lot of skill to retrieve the needle back between the main and lateral branch of the prepubic tendon. Although this technique appeared very promising, the entire procedure was to be performed blindly only by palpation in the cul-de-sac of vagina. In view of a limited bite in tlie vaginal floor and the prepubic tendon, the chances of rupture of the suture or the tearing of the vaginal wall could not be overlooked. Considering the complexity in execution, this technique is recommended only in the hands of an experienced worker. Button suture fixation was used on 8 cows. Commercially available suture needle (triangular and straight needle No.l) worked satisfactorily to put these sutures. Since the vaginal insertion of the suture was preplaced on the prolapsed mass, there was adequate visibility and a very little haemorrhage occurred in the placement of the suture. Amputation of prolapsed mass was easy in this technique as the entire mass was presented at the vulva for handling. However, due to the large size and irreparable changes in the vagina and the cervix, the wall of the organ was considerably thick making it difficult to cut. Submucosal resection of vagina was fairly simple than amputation. As the surgical dissection was restricted only to the submucosa of the exposed portion, haemorrhage from the dissection was comparatively less. Since the technique did not involve extensive dissection or ligation of blood vessels, the procedure could be completed in comparatively lesser time. In cases of per vaginum panhysterectomy, the dorsal wall of the vagina bled considerably. Further dissection for the uterine junction and ovarian stumps had to be done blindly by palpation with fingers. It was felt necessary to put two ligatures on the stump to ensure complete obliteration of the ovarian blood vessels. After having completed this procedure, small length of vagina was left behind so that there was no chance of recurrence of prolapse. Per vaginum bilateral ovariectomy was performed in the similar manner as in pervaginum panhysterectomy. The uterus and cervix were left intact. For performing the ovariectomy, a specially designed Richards' ovariotome was used to crush the ovarian blood vessels and to cut the pedicle. Suturing of the vaginal incision (colpotomy) did not pose major problem and the entire surgery could be completed in 20 to 30 minutes. In Group 1 and II, recurrence of prolapse was noted in one cow each due to rupture of sutures. In one more case of group II, pyometra was noted due to foreign body i.e. buttons. In Group III, all the animals showed mild to moderate bleeding from vagina .and mild straining. This was treated by local infusion of Betadine solution and application of Ceftriaxone powder. This bleeding disappeared towards 7-8 days and effectively stopped at 10 days. One cow showed recurrence of prolapse. In Group IV, minor haemorrhage through vagina was noted in all the cases for 1 or 2 post-operative days. All the animals recovered uneventfully, except one cow that suffered prolapse at 24th post-operative day. In Group V, all the animals, except one cow, showed slight haemorrhage for a period of 3 to 4 days followed by slight mucous discharge for next 3 to 4 days, but recovered uneventfully by 15 days. Prepubic tendon fixation and Button suture fixation were performed in those cows where the prolapse was mild to moderate type (Grade II) with no complications like oedema, necrosis, gangrene and other secondary complications like maggots infestation. As such these two techniques were useful in conserving the genitalia. However, button suture fixation method was preferred to address the clinical condition. Two cows treated with button fixation technique conceived subsequently. Thus, these animals would have become useful for reproduction in future. Submucmosal resection was comparatively easier to perform and resulted into less serious haemorrhage. In those cases where the changes in the prolapsed organ appeared irreversible, it was decided to remove entire reproductive tract per vaginum (Panhysterectomy). This surgical technique also was quite demanding owing to large number of engorged blood vessels in the dissection. Per vaginum bilateral ovariectomy was reserved for those cases where ovarian changes were marked. It was postulated that the prolapse was primarily due to ovarian dysfunction. This was proved when the ovariectomy was performed and the prolapse was repositioned. No recurrence was noted in any of the six cases until eight days. However, this procedure was performed in ox cases and only had moderate Grade II prolapse. Therefore it requires further study. Haemato-biochemical and serum endocrinological assessment paved a clinical way of understanding the etiopathology, stress and therapeutic resolution of this complex malady in cows by contemplating the findings to the clinical merits of the cases retrospectively. Various micro organisms were isolated from the vaginal swabs from these cows. Antibiotic sensitivity test indicated that Ceftriaxone and Enrofloxacin were both effective against these organisms while Amoxirum was not as effective. Urinalysis can be used as one indictor to assess pre-operative status and effect of surgical treatment. Histopathological studies of genital tissue on surgical removal in Group I and II, did not show significant lesions while Group III, IV and V cases revealed, variable degrees of inflammatory lesions, characterized by degeneration, edema necrosis and fibrosis. Moderate to severe infiltration of mononuclear cells in the vaginal and cervical mucosa were found in groups III and IV. Group V indicated necrosis of superficial mucosal lining with the presence of bacterial colonies. Hyperplasia of glands of vaginal and cervical mucosa showed neutrophils and mononuclear cells infiltration with cystic dilatation. The group V cases revealed chronic inflammatory changes of adhesions, lacerations, necrosis and perivascular fibrosis, hyperplasia of uterine glands along with congestion and haemorrhage. The group VI cases, (with abnormalities of ovaries) revealed cystic dilation with fluid filled cavity, with single layered cysts. Few specimens revealed multicystic ovaries which had thick, multilayered wall and cyst within the wall. The histological signs correlated with the clinical signs and helped in prognosis of repair and cure of the case.ThesisItem Open Access INFLUENCE OF DIFFERENT CATEGORIES OF FOLLICLES ON QUANTITY AND QUALITY OF OOCYTES WITH RESPECT TO THEIR IN VITRO MATURATION IN SURTI BUFFALO(AAU, Anand, 2011) SINGH, RAHUL PRATAP; Shah, R. G.The present study was conducted on buffalo abattoir ovaries at Laboratory of Biotechnology of Veterinary College, Anand. The study was conducted over nine months from February 2010 to October 2010 on 499 ovaries. They had 1740 different size of follicles; from them 1046 different grades of oocytes were recovered for the study by slicing method. The influence of different categories of follicles on quantity and quality of buffalo oocytes, its maturation, effect of presence or absence of CL on oocytes recovery rate and its quality and in vitro fertilization of matured oocytes were studied. The total numbers of follicles recovered from 499 ovaries were 1740, of them 726 follicles were observed on the ovaries which had all the three size of follicles. The overall mean number of follicles per ovary was found to be 2.40 ± 0.07. The mean number (percentage) of small, medium and large size follicles per ovary were 3.16 ± 0.11 (49.40), 1.81 ± 0.06 (34.20), and 1.30 ± 0.05 (16.40), respectively. Oocytes collected by slicing method were classified on the basis of cumulus investment and ooplasm homogenecity, viz. grade A (>3 layers of cumulus cells), grade B (1-3 layers of cumulus cells), grade C (less compact cumulus) and grade D (nude oocytes). The mean number of oocytes per ovary of grade A (2.92 ± 0.14) and B (2.49 ±0.13) were significantly higher (P<0.05) followed by grade C (1.66 ± 0.09) than that of grade D (1.32 ±0.10) oocytes. The correlation studies between follicles size and oocytes qualities indicated that the small (<5 mm) follicles had highly significant (P<0.01) and positive correlations with grade B and C oocytes. Similarly, medium size follicles (5-8 mm) showed highly significant (P<0.01) and positive correlations with grade A oocytes. Large follicles (>8 mm) showed significant (P<0.05) and posiive correlation with grade A oocytes, whereas it had negative correlation with grade D, C, and B oocytes. The maturation rate achieved was 80.97 per cent in TCM-199 supplemented with 0.6 per cent BSA. The highest percentages of cytoplasmic maturation observed in oocytes of grade A, B, C and D were for good (50.30 per cent), fair (37.70 per cent) and poor (33.30 per cent) and poor (28.60 per cent) quality of oocytes, respectively. The significantly higher number of grade A (>3 layers of cumulus cells) and grade B (1-3 layers of cumulus cells) quality of oocytes attained good cytoplasmic maturation than grade C (less compact cumulus cells) and grade D (nude oocytes). The total maturation rate from grade A oocytes was highest (87.94 per cent) followed by oocytes of grade B (82.91 per cent) and grade C (72.22 per cent). The nuclear maturation was evaluated in 528 oocytes by Hoechst 33342 stain. The highest number of grade A oocytes (26 per cent) reached to M-II stage, followed by grade B (24.21 per cent), and grade C (14.28 per cent) and none of the oocytes from grade D reached to M-II stage, they were mainly arrested at GV and GVBD stage. The grade D oocytes did not mature and maximum per centage of degenerated oocytes (72.50 per cent) was found in this category. Of the 367 ovaries, the CL was present on 73 and absent on 294 ovaries that yielded 74 and 972 oocytes, respectively. Significantly (P<0.05) greater number of oocytes per ovary were recovered (5.06 ± 0.36) when the CL was absent compared with ovaries on which CL was present (0.38 ± 0.05). Further, significantly higher percentage (P<0.01) of recovery rate of grade A (37.76 per cent) and grade B (37.96 per cent) oocytes was obtained from the ovaries in which CL was absent than the ovaries in which CL was present (grade A: 40.54 per cent and grade B: 39.19 per cent). The effect of presence Vs absence of CL on the ovaries revealed 8.11 Vs 18.00 per cent recovery rate for grade C and 12.16 and 6.27 per cent for grade D oocytes. Oocytes of grade A (n=181) and B (n=305), which had cytoplasmic maturation, were utilized for in vitro fertilization. The overall fertilization rate observed was 20.16 per cent for grade A and B occytes. The higher fertilization rate was observed for grade A (21.00 per cent) oocytes than that of grade B (19.67 per cent). Application of in vitro embryo production technology in assisted reproduction of buffalo will not only improve productive and reproductive potential of the buffalo population but will also help to rescue the precious germ plasma going to waste by indiscriminate slaughter of this animal. This study showed that functional structure on the ovaries, i.e. corpus luteum, size of follicles etc. affect the recovery rate and quality of oocytes. The higher oocyte recovery rate and good quality oocytes were observed in absence of corpus luteum on the ovaries. It also revealed that the presence of cumulus layer around the oocytes affect the maturation rate. Further studies required for the in vitro culture system for in vitro embryo production of buffaloes.ThesisItem Open Access STUDIES ON EFFECT OF SILYMARIN IN COMBATTING TOXICOPATHOLOGICAL EFFECT OF DOXORUBICIN IN WISTAR RATS(AAU, Anand, 2011) NEVASE, SUJIT NAMDEV; DAVE, C. J.The anthracycline group of antibiotics includes most effective classes of antineoplastic agents available for the treatment of human and animal cancers. Among the anthracyclines, Doxorubicin (DOX) is an important anticancer agent. It is one of the most valuable components of various chemotherapeutic regimens of human and animal cancers. Reactive oxygen species have been demonstrated to play an important role in doxorubicin induced toxicity. Silymarin, an extract from Silybum marianum has potentially beneficial antioxidant properties. The present study was designed to examine the alleviative effect of silymarin on doxorubicin induced toxicity in rats. A total of 48 rats were randomly divided into 4 different groups. Each group consisted of 6 male and 6 female rats. The groups were numbered as group I to IV. The group I served as control and received only vehicle (Coconut oil 1ml). Group II, received silymarin at doses of 200 mg/kg daily for 28 days orally. Group III received doxorubicin + silymarin .doxorubicin at doses of 3mg/kg on 1st, 7th, 14th, 21st and 28th day i/p and silymarin at doses of 200mg/kg daily orally for 28 days, and group IV received Doxorubicin alone at doses of 3mg/kg on 1st , 7th, 14th, 21st and 28th day i/p . After completion of 28 days treatment, blood samples were collected for hematology and serum biochemical analysis from retro-orbital plexus with the help of capillary tube. The animals were sacrificed by high dose of anesthesia with Di - ethyl ether. Necropsy examination was performed in all sacrificed animals and gross lesions were recorded. Tissue samples (lung, liver, kidney, intestine, testes and heart) were collected in 10% formalin solution for histopathological examination. Male and female rats of all the groups did not reveal any mortality upto the 28 days, after doxorubicin and silymarin administration. Signs like weakness, loss of appetite, diarrhoea and thickening of the skin at injection site and mild alopecia were noticed in group IV rats. The significant (P<0.05) reduction in body weight gain and feed consumption in both male and female rats of group IV (DOX alone) and group III (DOX+SILY) were observed. However, significant restoration (P<0.05) in body weight gain and feed consumption by silymarin in both male and female rats of group III (DOX+SILY) was observed as compared to group IV(DOX alone). The doxorubicin treated group showed significant decrease in relative organ weight of liver, heart and testes. However, treatment with Silymarin effectively reduced the doxorubicin induced alterations in relative organ weight in these organs. Anaemia, leucopaenia, hypoproteinaemia, hypoalbuminaemia, elevated serum ALT, AST, AKP and LDH levels, increased BUN and creatinine were observed in doxorubicin treatment group IV. While concurrent administration of silymarin with doxorubicin in group III reported significant protection (towards normal) against doxorubicin induced alteration in blood cells and serological value. The histopathological alterations observed in different organs viz., liver, kidney, lungs, heart and testis in group IV (DOX alone) were of sever degree. Liver of rats from group IV showed vacuolar degeneration of hepatocyte, dilatation of central vein and sinusoids and pronounced scattered haemorrhages with distortion of hepatic cords. Kidney of rats from group IV revealed tubular necrosis, large hyaline casts in tubular lumen, desquamation and cloudy swelling of the tubules accompanied by degeneration, tubular dilatation, glomerular congestion and inter-tubular haemorrhages in the kidney. Heart of Rats from group IV showed disrupted cardiac muscle fibres, interfibrillar congestion, haemorrhage and moderate infiltration and testes revealed atrophy of seminiferous tubule, vacuolation. Shrunken seminiferous tubule showed loss of germinal epithelium and widening of interstitial spaces. However, group III exposed to doxorubicin with silymarin revealed only mild type of histopathological alterations in all organs in comparison to group IV, showed restoration of histological alterations towards normal. These results have suggested that, silymarin is effective in combatting doxorubicin induced toxicopathological effect in wistar rats.