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ThesisItem Open Access EXPRESSION PROFILING OF MICRORNA IN LIPOPOLYSACCHARIDE CHALLENGED PERIPHERAL BLOOD MONONUCLEAR CELLS OF CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) AKSHARA B. VENU; Dr. Divya P.DMicroRNAs are endogenous, small non-coding RNAs approximately 20-22 nucleotides long which play a key role in gene regulation by affecting translation and transcription mechanisms. The present study was undertaken to analyse the differences in the microRNA (miRNA) expression in (LPS) challenged peripheral blood mononuclear cells (PBMCs) of cattle. The current research work was carried out using the isolated blood samples from apparently healthy female crossbred cattle maintained at the University Livestock Farm and Fodder Research and Development Scheme, Mannuthy. Isolation of PBMCs were done by density gradient centrifugation using HisepTM lymphocyte separation medium 1077.Expression of miRNAs viz; bta-miR-451, bta-miR-107, bta-miR-93 and bta-miR-125a, whose expression was found to be varying in the LPS treated and untreated bovine PBMCs by miRNAome analysis of previous studies was validated in the present study by qRT-PCR assay. Insilico analysis was done using various online tools for the prediction of target genes of real time PCR validated miRNAs, gene ontology analysis and also to study the cellular pathways associated to the target genes. Targets of each miRNAs were predicted using the online target prediction programme TargetScan Human 8.0. Through the use of the online DAVID bioinformatics tool, gene ontology analysis of the real time PCR validated miRNAs was carried out. Predicted targets for each miRNAs were annotated to biological process, molecular function and cellular component categories. The pathway analysis programme (KEGG) of the DAVID database was utilized to analyze and interpret the pathways associated with the predicted targets of real time PCR verified miRNAs. The study also included the assessment of cytokines associated to the cellular pathways influenced by the target genes of real time PCR validated miRNAs in the supernatants of PBMC cultures from both treatment and control groups by ELISA. The expression of miRNAs; bta-miR-451and bta-miR-107 was found to up regulated while bta-miR-93 and bta-miR-125a showed decrease in their expression in LPS treated PBMCs when compared to control cells. The results of real time PCR validation of aforementioned miRNAs expression were in accordance with the findings of miRNAome analysis. Besides, significant enrichment of target genes of the real time validated miRNAs was noticed in many immune related GO terms as well as in critical immune associated cellular pathways. Measurement of cytokine, TNF α revealed a significantly higher level in LPS treated PBMCs when compared to LPS untreated cells. The findings of the current study will help in understanding the role of miRNAs in LPS mediated immune responses in cattle.ThesisItem Open Access SEMEN QUALITY AND PRESERVABILITY IN ASEEL, KADAKNATH AND NATIVE CHICKEN OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) AKSHAY D; Dr. Harikrishnan SAn experiment was conducted at Department of Poultry Science, College of Veterinary and Animal Sciences, Mannuthy to evaluate the semen quality and preservability in twenty-four adult roosters of 30 of weeks age (eight each from Aseel, Kadaknath and native chicken of Kerala). The birds were housed in individual cages with dimension of 39 cm × 46 cm × 44 cm and maintained with a diet containing 16 per cent crude protein, 2600 Metabolizable Energy / kg diet and ad-libitum water. The semen was collected once in four days by the abdominal massage technique during the experimental period.In the first phase of study, average of three observations on microscopic and macroscopic parameters of semen and hypo-osmotic sperm swelling response wereused to select best three birds from each breed. In second phase of study, pooled semen samples from each selected breeds were used to evaluate heat shock resistance, cold shock resistance and storage study. In storage study, the semen samples were diluted with four different extenders viz. Normal Saline (NS), Phosphate Buffer Saline (PBS), Lake Poultry Semen Extender (LPSE) and Beltsville Poultry Semen Extender (BPSE) kept at room and refrigeration temperatures (4°C) and the progressive motility after 0, 2, 4, 8, 16, 24 and 48 h was evaluated. The study was replicated six times.The semen volume in native chicken of Kerala was significantly (p<0.05) higher compared to other two indigenous breeds. Most of the collected semen samples were white in colour [Aseel (80.95 per cent), Kadaknath (90.48 per cent) and native chicken of Kerala (91.67 per cent)]. Regarding the semen consistency, Kadaknath and native chicken of Kerala had a thick semen while thick with slight watery semen was observed in Aseel roosters. Significantly (p<0.05) higherappearance score was observed in native chicken of Kerala and Kadaknath while lower for Aseel roosters. The major semen contaminant observed in all the indigenous breeds was faeces followed by blood and uric acid crystals. The semen pH was found to be slightly alkaline in all the three breeds. There was no differencein the sperm density among the three breeds. Significantly (p<0.05) higher mass activity score was measured in native chicken of Kerala and lower for Aseel roosters while a comparable value was observed in Kadaknath roosters. The native chicken of Kerala had significantly (p<0.01) higher initial progressive motility and lower for Aseel, while results were comparable in Kadaknath roosters. The per cent of live sperms were significantly (p<0.01) higher in native chicken of Kerala than Kadaknath and Aseel roosters. There was no statistical significance in HOS reactive spermatozoa between the three breeds. Better heat shock and cold shock resistance was exhibited by Kadaknath breed. However, native chicken of Kerala was also in par with Kadaknath in case of heat shock resistance.After eight hours of storage at room temperature, Aseel and Kadaknathsemen diluted with LPSE exhibited a better progressive sperm motility while for native chicken of Kerala, both LPSE and BPSE extenders had good results. The preservability in PBS and NS were significantly (p<0.01) lower compared to LPSE and BPSE at room temperature in all the three breeds. At refrigeration temperature,LPSE was superior in retaining the progressive motility followed by BPSE. The progressive motility was zero in the semen samples diluted with NS among all the indigenous breeds at 48 h of storage. The native chicken of Kerala alone showed a feeble progressive motility in PBS at 48 h of storage.To conclude, the semen volume, appearance score, mass activity score initial progressive motility and sperm viability was higher in native chicken of Kerala compared to Aseel and Kadaknath. The sperm density, abnormality of sperms and HOS response were comparable in all the three indigenous breeds. The Kadaknath breed had significantly better heat shock and cold shock resistance. The native chicken of Kerala was also comparable to Kadaknath breed in terms of heat shock resistance. Comparing the effectiveness of various poultry semen extenders, LPSE was found to be superior in maintaining the sperm motility at room and refrigeration temperature followed by BPSE. The sperm viability has increased up to six times as a result of semen preservation in refrigeration temperature.ThesisItem Open Access EFFECT OF REPEATED FREEZING AND THAWING ON THE QUALITY OF SPENT CATTLE AND BUFFALO MEAT(COLLEGE OF VETERINARY & ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) HRIDHYA VIJAY C; Dr.Irshad AThe objective of the present study was to assess the effect of repeated freeze-thaw cycles on the physic-chemical, microbial, nutritional and sensory qualities of spent cattle (n=6) and buffalo meat (n=6). The effect of three successive freeze-thaw cycles on beef and carabeef was compared with raw meat (before freezing). The biceps femoris muscle was collected and subjected to freezing at -18 ℃ for seven days, after ageing for 72 hours. The samples were thawed by five different methods, viz., ambient temperature thawing (AT) at 31-34°C, chiller temperature thawing (CT) at 4 °C, microwave thawing (MT), immersion thawing with water replacement (ITW) and immersion thawing without water replacement (ITWO). All the meat samples were subjected to three freezing (one week) and thawing cycles. The post-thaw meat after each freezethaw cycle was subjected to physico-chemical, microbiological, nutritional loss and sensory attribute assessment. The results of the present study suggested that as the number of freeze-thaw cycles increased, physico-chemical parameters such as pH, water holding capacity, shear force value, moisture content and redness (a*) value declined significantly (P≤0.05). On the other hand, the thaw loss, MFI, Lightness (L*) and yellowness (b*) value, thiobarbituric acid value, total ash and collagen solubility increased significantly (P≤0.05) and significantly differed between the thawing methods within the freeze-thaw cycles. The drip loss and tyrosine value did not differed significantly among the freeze-thaw cycle as well as among the thawing methods but an increase was observed in the 3rd freezethaw cycle compared to raw meat. Collagen content, cooking loss, crude protein and ether extract remained non-significant throughout the three freeze-thaw cycles and were not affected by thawing methods. Sensory characteristics like colour, odour, appearance, flavour, juiciness and overall acceptability declined significantly (P≤0.05), but tenderness increased, and it was well within the acceptable range as evaluated by the semi-trained panellists. There were significant differences (P≤0.05) observed between the thawing methods within the freeze-thaw cycles for the sensory attributes and CT had the best scores among the thawing methods in all the freeze-thaw cycles. Microbial quality was improved with successive freeze-thaw cycles. The total viable count increased initially in the first freeze-thaw cycle and then decreased gradually with an increase in freeze-thaw cycles without significant differences. Among the thawing methods, CT had the highest sensory scores and the least effects on physico chemical attributes. Therefore, based on the results of the present study, it can be concluded that as the number of freeze-thaw cycles increased, the physicochemical, microbial, nutritional, and sensory parameters were affected significantly and repeated freeze-thaw cycles should be minimised at processor as well as at consumer level as much as possible. It was also concluded that the best thawing method was CT followed by MT, ITWO, ITW and AT.ThesisItem Open Access EVALUATION OF OXIDATIVE STRESS IN STAGE III CHRONIC KIDNEY DISEASE IN DOGS AND ITS MANAGEMENT(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) NEETHU BALAKRISHNAN; Dr. Madhavan Unny N.Oxidative stress is considered to be one of the important contributing factors in progression of chronic kidney disease (CKD). The present study entitled “Evaluation of oxidative stress in stage III chronic kidney disease in dogs and its management” was conducted to assess oxidative stress and its management with N-acetyl cysteine (NAC) in stage III CKD dogs. Twelve dogs diagnosed with stage III CKD as per the guidelines of International Renal Interest Society were selected and divided into two groups of six animals each. The selected animals were subjected to detailed clinical examination, blood pressure measurement, urinalysis, ultrasonographic examination and assessment of haemato-biochemical and oxidative stress parameters on the day of presentation. Animals belonged to group I and II were subjected to standard therapy for 15 days. In addition to standard treatment, animals of group II were administered with NAC. Haemato-biochemical parameters, oxidative stress parameters, urinalysis and blood pressure were rechecked on 15th day of treatment. Highest incidence of CKD was observed in dogs aged between 6-12 years. Dehydration, polyuria, polydipsia, melena, vomiting and anorexia were the major clinical findings. Ultrasonographic changes recorded were hyperechogenicity, increased cortical thickening and poor cortico medullary distinction. Clinical response was moreevident in animals of group II compared to group I. At the end of the study period, a significant decline in lipid peroxidation level and total antioxidant status and significant increase in glutathione peroxidase level was recorded in group II. N-acetyl cysteine therapy in conjunction with standard treatment was found to be more effective than standard therapy alone in management of oxidative stress and thereby delaying progression of disease and increasing the survival time in CKD dogs.ThesisItem Open Access OCCURRENCE OF ENROFLOXACIN AND TETRACYCLINE RESISTANT KLEBSIELLA PNEUMONIAE AND SALMONELLA SPP. IN BEEF AND COW MILK(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY,THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-22) JISNA JAMAL P. P; Dr. C. LathaAntimicrobial resistance (AMR) is a global public health threat thatchallenges the scope and ability of modern medicine to effectively treat common bacterial infections. The present study was conducted for the isolation and identification of enrofloxacin and tetracycline resistant K. pneumoniae and Salmonella spp. from beef and raw cow milk samples. A total of 200 beef and 200 raw cow milk samples were collected from different retail outlets and milk societies of Thrissur and Palakkad districts. The samples were collected during a span of 10 months, from January 2022 to October 2022. The samples were subjected to conventional culture techniques followed by biochemical tests for the preliminary identification of the organisms. On analysis of milk samples from both districts, highest number of K. pneumoniae was obtained from Palakkad (86 per cent) district, followed by Thrissur (69 per cent) district. In the case of beef samples, 45 per cent and 30 per cent revealed the presence of K. pneumoniae from Thrissur and Palakkad districts, respectively. On statistical analysis, it was revealed that, the difference in occurrence of K. pneumoniae in milk samples from Thrissur and Palakkad districts were significant (p<0.01). Similarly, there was significant difference (p<0.05) between the occurrence of K. pneumoniae in beef samples from Thrissur and Palakkad districts. Among the 200 milk samples analysed, seven per cent of the samples from Palakkad district and five per cent of samples from Thrissur district were contaminated with Salmonella spp. A higher amount of Salmonella spp. was detected in beef samples from Thrissur (17 per cent) and Palakkad (12 per cent) districts. Statistical analysis revealed that there was no significant difference (p>0.05) in occurrence of Salmonella spp. in both milk and beef samples from Thrissur and Palakkad districts. On molecular confirmation, 91.03 per cent K. pneumoniae and all theSalmonella spp. isolates generated amplicons specific for rpoB and invA genes, respectively by PCR. None of the Salmonella spp. isolates in this study amplified virulence specific spvC gene on PCR. The confirmed isolates were subjected to phenotypic confirmation of antibiotic resistance by Kirby-Bauer disc diffusion test against 15 commonly used antibiotics. Multiple antimicrobial resistance (MAR) index was calculated for both K. pneumoniae and Salmonella spp. The MAR index for K. pneumoniae isolates analysed in this study ranged from 0.06 to 0.6, and that of Salmonella spp. showed a range of 0.06 to 0.33. The phenotypically enrofloxacin resistant isolates were subjected to Mismatch Amplification Mutation Assay (MAMA) PCR, to detect point mutations in gyrA and parC genes. The gyrAmutations were detected in 13 isolates of K. pneumoniae and three isolates of Salmonella spp. isolates. None of the isolates in this study showed parC mutations on MAMA PCR. The enrofloxacin resistant isolates were also screened for the presence of plasmid mediated quinolone resistant, qnrS gene. A total of 31 isolates, comprising 23 isolates of K. pneumoniae and eight isolates of Salmonella spp. amplified qnrS gene on PCR. Genotypic confirmations of tetracycline resistance in the isolates were done by targeting the tetracycline resistant genes tetA, tetB, tetO and tetX on PCR. The presence of tetA gene was detected in 58.7 per cent of K. pneumoniae isolates and 80 per cent of Salmonella spp. isolates. The gene tetB was detected in 43.48 per cent and 50 per cent of K. pneumoniae and Salmonella spp. organisms respectively. None of the isolates amplified tetO and tetX genes on PCR. The representative amplicons of the resistant genes were sequenced and submitted to National Center for Biotechnology Information (NCBI), and accession numbers were obtained. The results of the study revealed that beef and milk acts as an important reservoir of enrofloxacin and tetracycline resistant K. pneumoniae and Salmonella spp. This highlights an urgent need for the implementation of control strategies in management of food animals and handling of food products. In order to effectively control the emergence and spread of the resistance, inter-sectoral collaboration and a One Health Approach incorporating agriculture, animal husbandry, veterinary medicine, human medicine and environment science should be adopted. The use of antimicrobials in human and animals should be monitored and specific control policies which acknowledge the use of antimicrobials in food producing animals should be developed.ThesisItem Open Access MEDICAL MANAGEMENT OF CANINE PYOMETRA WITH PROSTAGLANDIN E1 ANALOGUE AND PROGESTERONE RECEPTOR ANTAGONIST(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-08-10) ARYA KRISHNAN S; Dr. Hiron M. HarshanA study was undertaken to evaluate the efficacy of different therapeutic protocols in the treatment of canine pyometra based on clinical, haemato biochemical, B-mode ultrasonographic and Doppler ultrasonographic evaluation. Twenty two bitches presented to TVCC, Mannuthy and UVH, Kokkalai and confirmed with pyometra were randomly grouped into three based on the treatment followed, as group I (mifepristone @ 5 mg/kg b.wt. PO b.i.d., misoprostol @ 10 mcg/kg b.wt. PO b.i.d.), group II (mifepristone @ 5 mg/ kg b.wt b.i.d., misoprostol @ 3mcg/kg b.wt. intravaginally b.i.d.), group III (mifepristone @ 5 mg/kg b.wt. PO b.i.d.). In bitches with serum progesterone > 2ng/mL on the day of presentation cabergoline was administered @ 5 mg/kg b.wt. PO o.d. for nine days. Haematology recorded anaemia, leucocytosis with neutrophilia in patients with pyometra, which resolved after treatment. Bitches treated withmifepristone and misoprostol (oral/ vaginal) attained a faster return to normal leucogram. Serological analysis revealed elevated progesterone, alkaline phosphatase and total protein, which reduced to physiological values by day 15 in animals of all the treatment groups. No abnormal values were marked in alanine transaminase, blood urea nitrogen or creatinine in all the bitches during the duration of treatment, probably as a result of excluding pyometra affected cases from medical management, which had gross systemic affections. B-mode ultrasonography revealed distended uterine horns with anechoic to hypoechoic contents and thickened wall in all bitches, which had reduced at statistically similar rate to normal by day 15 of treatment. Bacteriological isolates from anterior vagina revealed Gram negative bacilli followed by Gram positive cocci and Gram negative coccobacilli. Maximum sensitivity was noticed to Gentamicin, followed by cotrimoxazole and Ceftriaxone tazobactum in antibiogram. Resistive index was found to be a reliable tool as a recovery marker, as they increased from values below the marker values of 0.72 to above as the treatment progressed. RI values increased to 0.72 by day 3 in bitches treated with intravaginal misoprostol. Though bitches in all the groups attained recovery, in terms of haemato-biochemical and ultrasonographic observations a faster recovery rate was recorded in bitches treated with mifepristone and misoprostol (oral or intra vgainal). Based on clinical improvement, the bitches in group II had a mean recovery time of 2.42 + 0.48 days, which was significantly lower than that of bitches in group I and III. It could be concluded that all three protocols were effective for medical management of pyometra in bitches. The combination of oral misoprostol and intravaginal misoprostol along with supportive therapy was better in terms of clinical recovery.ThesisItem Open Access FORMULATION AND EVALUATION OF COMPLETE DRY FOOD FOR MEDIUM SIZED DOGS WITH MODERATE ACTIVITY(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) AKHIL PREM; Dr. Ally KAn experiment was conducted in medium sized adult nondescript dogs kept restrained for a period of four months to evaluate the formulated dry food. Four medium sized healthy adult nondescript dogs of about one to four years of age and around 10 to 15 kg body weight were selected and experimental design adopted was switch over design. The dietary treatments are T1 (40 per cent CP, 23 per cent fat, 30 per cent carbohydrate and 3626 kcal ME), T2 (35 per cent CP, 20 per cent fat, 38 per cent carbohydrate and 3528 kcal ME) T3 (26 per cent CP, 15 per cent fat, 52 per cent carbohydrate and 3475 kcal ME) and T4 (20 per cent CP, 11 per cent fat 62 per cent carbohydrate and 3370 kcal ME). The animals are allotted to each dietary treatment for a period of 4 weeks, one week was given in between the treatment to nullify the carry over effect wherein control diet was given following which animals are switched over to next dietary treatment. The blood samples were collected at the end of each trial period to estimate haemato-biochemical parameters. A digestibility trial was conducted at the end of each trial period for three consecutive days to determine the digestibility of nutrients.The average initial body weights of dogs in the T1, T2, T3 and T4 groups were 10.38, 10.40, 13.42, and 11.62 kg respectively and the corresponding values after feeding trial were 10.38, 11.60, 13.46 and 11.64 kg respectively. The average body score of dogs initially in the T1, T2, T3 and T4 groups were 4.00 ± 0.04, 4.05 ± 0.03, 4.23 ± 0.03 and 4.00 ± 0.07, respectively and the final body score at the end of feeding trial were 4.00 ± 0.07, 4.00 ± 0.04, 4.25 ± 0.03 and 4.00 ± 0.04, respectively in T1, T2, T3 and T4 groups. The average daily dry matter consumption of dogs maintained on four experimental diets were 151.23, 158.52, 164.06 and 173.02g for T1, T2, T3 and T4 respectively. Average DMI per kg body weight were 12.94, 13.93, 14.27 and 14.88g for dogs in T1, T2, T3 and T4 groups respectively. The average daily DMI per 100 kg body weight were 1293.73, 1392.65, 1426.53 and 1488g and average daily DMI per kg metabolic body weight were 23.9, 25.55, 26.25 and 27.46 g, respectively for the animals in the four treatment groups (T1, T2, T3 and T4). The data on average daily crude protein consumption of dogs maintained on the four experimental rations were 60.14, 55.48, 42.7 and 34.88g, respectively. The average CP intake per kg body weight was 5.14, 4.87, 3.71 and 3.00g for animals in T1, T2, T3 and T4 groups, respectively. The weekly average daily CP intake per 100 kg body weight was 0.51, 0.48, 0.37 and 0.29 kg and average daily CP intake per kg metabolic body weight was 9.50, 8.94, 6.83 and 5.54 kg, respectively, for T1, T2, T3 and T4 groups. The digestibility coefficient of nutrients in the four experimental rations (T1, T2, T3 and T4) were 79.39, 77.23, 74.83 and 72.26 per cent for dry matter, 82.92, 80.48, 85.26 and 82.34 per cent for crude protein, 95.99, 95.66, 97.15 and 95.18 per cent for ether extract, 48.34, 47.20, 47.98 and 46.17 per cent for crude fibre and 59.16, 60.59, 60.47 and 58.71 per cent for nitrogen free extract, respectively. The mean daily faecal output of the experimental animals maintained in T1, T2, T3 and T4 during digestibility trial are 119.37, 137.85, 171.55 and 188.06g respectively. The faecal output per kg body weight was 10.17, 11.97, 14.78 and 16.15g, resectively in T1, T2, T3 and T4. The faecal score of animals maintained in T1, T2, T3 and T4 groups were 3.10, 2.75, 2.48 and 1.95, respectively. The calculated value for total digestible nutrients (TDN) 98.47, 92.42, 85.47 and 75.80 per cent, digestible energy (DE) 4341, 4074, 3768 and 3342 kcal/kg feed and metabolizable energy (ME) 3559, 3340, 3089 and 2740 kcal/kg feed for the four diets T1, T2, T3 and T4, respectively. The average daily ME intake was 783.37, 799.18, 814.45 and 832.97 kcal. The average daily ME intake per kg metabolic body weight was 123.82, 128.82, 130.30 and 132.17 kcal for animals in T1, T2, T3 and T4 groups respectively.The values of various haematological and serum biochemical parameters estimated in T1, T2, T3 and T4 were 15.9, 15.73, 15.43 and 15.9g/dL haemoglobin, 43.35, 42.7, 42.45 and 41.38 per cent packed cell volume (PCV), 7.02, 7.08, 6.89 and 7.03 red blood cell (106), 10.38, 9.83, 11.18 and 10 white blood cell (106), 22.27, 21.93, 23.53 and 21.44 blood urea nitrogen (mg/dL), 2.62, 2.71, 2.81 and 2.61 g/dL serum albumin, 6.88, 6.42, 7.05, 6.81 g/dL serum total protein and 65.38, 66.02, 65.87 and 68.46 IU/L serum alkaline phosphatase (ALP), respectively. The overall acceptability of four experimental rations T1, T2, T3 and T4 are 5.7, 6.4, 6.8 and 6.2 respectively. The T3 experimental diet was ranked as the most acceptable followed by T2, then T4 followed by T1 which was ranked as least acceptable among the four experimental rations. Total cost of feed and cost of feed per kg were Rs.6573.2, 4781.1, 2693.2, 1483.3 and Rs. 329.65, 231.31, 127.28, 66.46 respectively for T1, T2, T3 and T4 feeds. All the experimental rations met the requirements for metabolizable energy and crude protein intake as stipulated by NRC (2006) and ICAR (2013).Based on the observations, the experimental rations T3 and T4 are found to be more acceptable. The carbohydrate content in T4 was higher than T3. Also T3 performed better than T4 in case of faecal scoring and consumer acceptance. So it can be recommended that pet food for medium sized dogs with moderate activity in Indian conditions can be made with 26 per cent protein, 15 per cent fat and 52 per cent carbohydrate.ThesisItem Open Access HISTOMORPHOLOGICAL EVALUATION OF STROMAL COLLAGEN AND IMMUNOHISTOCHEMICAL ANALYSIS OF HEAT SHOCK PROTEIN 47 IN CANINE MAMMARY TUMOURS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-22) KEERTHANA SANTHOSH; Dr. Devi S.SThe present study was conducted to histomorphologically evaluate the stromal collagen and to analyse the immunohistochemical localisation of Heat shock protein 47 (Hsp47) in canine mammary tumours (CMTs). Twenty five CMT samples were collected from Department of Surgery and Radiology, TVCC, Mannuthy and University Veterinary Hospital, Kokkalai. The highest occurrence of mammary tumour was observed in dogs of nine to eleven years age group and the mean age of occurrence of CMTs in the studied population was 9.04±0.54 years. Out of 25 CMTs, the highest occurrence was observed among female dogs. Data on breed-wise occurrence showed that Labrador retriever breeds were mostly affected. Grossly CMTs varied in shape, colour and consistency with a mean size of 10.34±0.91 cm. Based on histological features, 24 tumours were classified as malignant and only one as benign. Twenty four malignant CMTs were subjected to grading on the basis of tubule formation, nuclear pleomorphism and mitotic figures.Grade II tumours were high in number which was followed by Grade III tumours. Tumours were histologically classified into different subtypes and ductal carcinomas were high in number followed by solid carcinomas and tubulopapillary carcinomas. Tumour to stroma ratio (TSR) was assessed in H and E stained sections and immunohistochemically using anti-cytokeratin antibody and subsequently classified tumours into those with high TSR (low stroma), moderate TSR (moderate stroma) and low TSR (high stroma). Tumours with high and moderate stroma were further analysed and based on the extent of fibroplasia, stroma was classified into three: stroma with severe fibroplasia, stroma with moderate fibroplasia and stroma with mild fibroplasia. Nine CMT samples had severe fibroplasia, 11 had moderate fibroplasia and only two had mild fibroplasia. Collagen in the stroma was visualized by employing several staining techniques. Picrosirius red stain imparted red colour to collagen whereas Masson’s trichrome gave blue colour to collagen. Varying grades of collagen deposition could be appreciated in the stroma of cancerous tissue. With Herovici staining newly deposited young and immature collagen in the stroma of CMTs was identified. The sequences of changes occurring in collagen fibres during the progression from moderately differentiated CMTs to poorly differentiated ones were assessed in picrosirius red stained histological sections using polarized microscopy. In moderately differentiated tumours, thick yellow orange birefringence was noticed. In poorly differentiated tumours, a transition from yellow orange to greenish yellow birefringence was seen. Immunohistochemistry was performed to study the expression of Hsp47 in CMTs. Out of 25 tumour cases, 19 (seventy six per cent) were strongly positive, four (sixteen per cent) were moderately positive and two (eight per cent) had weak immunopositivity. Analysis of data revealed that IHC scores of Hsp47 positively correlated with tumour grades and all tumours with high fibroplasia showed strong immunopositivity for the protein indicating a clear association between the stromal fibroplasia and Hsp47. The deposition of collagen type I in CMTs having high and moderate fibroplasia was analysed immunohistochemically using antibodies directed against COL1A1. The results revealed that tumours having high and moderate stromal fibroplasia gave either very strong or strong immunostaining for the protein indicating an increased deposition of collagen type I in neoplasms of canine mammary glands. Statistical analysis revealed a strong correlation between COL1A1 expression and Hsp47 expression. The study proved that Hsp47 directly enhanced fibroplasia and favoured collagen deposition. Increased immunostaining of these two proteins corresponded to increase in grades of CMTs. Further studies in a larger population would help in confirming the potential roles of Hsp47 and COL1A1 as prognostic and therapeutic biomarkers in CMTs.ThesisItem Open Access IN VIVO EFFICACY OF GARLIC AND CHITOSAN FOR CONTROL OF CAMPYLOBACTER SPP. IN MEAT TYPE CHICKEN(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-17) KAVYA A; Dr. Deepa JollyCampylobacteriosis, a major foodborne gastroenteritis in humans, is acquired primarily through the consumption of contaminated poultry meat. Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the most frequently reported thermophilic Campylobacter spp. The present study was undertaken to assess the occurrence of Campylobacter spp. in meat type breeds by conventional and molecular techniques and to assess the in vivo anticampylobacter effect of garlic and chitosan in meat type chicken. A total of 120 cloacal swabs of different meat type breeds from the University Poultry and Duck Farm (UPDF), Mannuthy were subjected to isolation and identification of Campylobacter spp. byconventional culture techniques. Isolates were confirmed by multiplex Polymerase Chain Reaction for genus and species identification. The overall occurrence of Campylobacter spp. in the farm was 31.67 per cent with no significant difference in the occurrence in different meat type breeds. Of the isolates, 71.05 per cent were identified as C. jejuni and 28.95 per cent as C. coli. Two natural compounds viz., garlic [Fresh Garlic Extract (FGE) and Garlic Powder (GP)] and chitosan were analysed for in vitro anticampylobacter activity by determining the Minimum Inhibitory Concentration (MIC).The Standard strain C. jejuni NCTC 11168 and one of the confirmed C. jejuni isolate (wild strain) were found to be more susceptible to chitosan than garlic. In vivo study was undertaken in UPDF, Mannuthy with 78 day-old meat type chicks. Oral challenge was performed on the third day with the wild isolate of C. jejuni and six birds were randomly examined for Campylobacter colonisation, which revealed cent per cent colonisation on 20th day. Four treatments (groups of 12 birds) which included, T1-feed+ground fresh garlic, T2-feed+GP, T3-feed+chitosan, T4-feed+chitosan+GP, alongwith positive and negative controls, were initiated on 21st day. After first and second weeks of treatment, statistical analysis revealed a significant reduction in Campylobacter count in all treatment groups with the lowest reduction in T3 group. None of the treatments could completely eliminate the organism. Histopathological examination showed degeneration and shortening of villi in the caeca and inflammatory changes in the liver. Antibiotics resistance profile of the isolates against 14 commonly usedantibiotics revealed the presence of multidrug-resistant Campylobacter and all the isolates showed complete resistance to cefotaxime. The Study highlighted the importance of preharvest strategies in farms along with strict biosecurity and hygienic measures to reduce the Campylobacter load in poultry.
