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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access LACTATION PERFORMANCE AND MILK COMPOSITION OF ATTAPPADY BLACK GOATS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-08-30) SUDHARSAN M.; A. KannanThesisItem Open Access PORCINE DERIVED SCAFFOLD ASSISTED FULL THICKNESS SKIN WOUND HEALING IN RABBIT MODEL(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-06-15) SUVANEETH P; SUVANEETH P; N. Divakaran Nair; N. Divakaran NairThe remodeling and regenerative responses of porcine cholecyst on full thickness skin wounds were evaluated in this study using rabbit as an animal model. Comparison of porcine cholecyst, porcine cholecyst seeded with autologous bone marrow cells and open wound control were made for evaluation. Porcine cholecyst was decellularized using a non-enzymatic, non-detergent based protocol and was checked for cellularity prior to in vivo evaluation. The in vivo evaluations were done on full thickness skin wound healing model in New Zealand White rabbits. The inflammatory, remodeling, and regenerative responses were evaluated 7, 14, 21 and 28 days post implantation. The H&E stained sections were evaluated for inflammatory and remodeling responses. Collagenization was evaluated and quantified using Masson’s trichrome and Picrosirius red staining. Differential collagenization was quantified using Herovici staining. Elastin deposition was ascertained using Verhoeff-Van Gieson’s staining. Proliferative response of the fibroblasts was evaluated by argyrophilic nucleolar organizer region (AgNOR) histochemistry and proliferating cell nuclear antigen (PCNA) immunostaining. Dermal cellular proliferation was quantified using PCNA immunohistochemistry. Re-epithelialization and epidermal formation was quantified using cytokeratin based immunohistochemistry. Vimentin immunohistochemistry was used to assess the mesenchymal cell response. ASMA immunohistochemistry was carried out to assess myofibroblast activity and CD 31 for neoangiogenesis. The results of the current study indicated that porcine derived cholecyst scaffolds are very well suited to be used as a bioscaffold material for full thickness skin wound healing, owing to their biochemical, biodegradable, biocompatible and tissue remodeling responses. The use of porcine cholecyst in full thickness skin wounds showed improved epithelialization and faster remodeling devoid of infections or graft rejections in all the animals under study. Scab formation, ulcerations, infections and other complications were also not observed in any of the scaffold assisted wounds. The scaffold enhanced cellular proliferation and keratinocyte activity. Controlled collagenization and faster replacement of immature collagen to mature collagen were also observed in scaffold assisted treatments. Improved neoangiogenesis were seen on scaffold assisted wounds from day seven itself which aided in faster healing of granulation tissue. Complete healing occurred around 14 days in graft assisted treatments, which was significantly early for the size of the wounds. Porcine cholecyst assisted healed wounds showed minimal wound contraction and reduced the chances of scar formation. The presence of autologous marrow cells enhanced proliferating cells, possibly due to the presence of bone marrow derived mesenchymal stem cells, as observed by a higher mesenchymal cell activity in vivo in marrow cells supplied scaffold. Use of autologous marrow cells has improved the time of healing in porcine cholecyst assisted skin wound healing, but not significantly from the application of a non-cell seeded matrix.ThesisItem Open Access DEVELOPMENT OF A MODIFIED INACTIVATED VACCINE AND ITS COMBINATION WITH A RECOMBINANT PROTEIN AGAINST LEPTOSPIROSIS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-12-30) MANJU SOMAN; M.MiniThe study was taken up with an aim to develop a foolproof technique for prevention of leptospirosis. It involved the development and immunity evaluation of a modified whole cell inactivated vaccine incorporating the predominant leptospiral serovars, Australis, Autumnalis and Pomona in hamsters. The study also ascertained the genus specific immunoreactivity of a truncated recombinant leptospiral LigA protein and its combination with the modified inactivated vaccine, in hamsters. The immunomodulatory effect of incomplete Freund’s adjuvant (IFA) and the aluminium hydroxide gel adjuvant in hamsters was also assessed. Primers were designed for a highly immunodominant region of the ligA gene, spanning nucleotides from 1873 to 3363. The PCR amplified 1491 bp fragment of ligA DNA was cloned into pET -32a vector and expressed in E.coli BL21(DE3). The conditions optimum for expression of this recombinant protein were analysed. Maximum expression was obtained following induction with 2 mM IPTG, at an incubation temperature of 28o C following six hours of incubation at 200 rpm shaking speed. The Ni-NTA purified rLigA protein was used for immunization of hamsters. The optimum concentration of the rLigA protein and modified inactivated vaccine required for immunisation of hamsters, was determined by immunising four sets of hamsters with four different concentrations of the antigens, 14 days apart. It was revealed that the concentration of 80µg/ 40 µg of Lig A protein and 108 leptospires per millilitre gave the maximum IgG ELISA and MAT titres.Six vaccine groups were set up for six different vaccine combinations which included the modified inactivated vaccine, rLigA protein and a combination of the modified inactivated vaccine and rLigA protein. Adjuvants IFA and aluminium hydroxide were used in the study. The serum antibody titres on days 0, 7,14 and 27 were determined by MAT and recombinant IgG ELISA The virulence of laboratory strains of Leptospira interrogans serovars Pomona (homologous) and Icterohaemorrhagiae (heterologous) was enhanced by serial passage in hamsters and these were used as challenge organisms in the study. The LD50, of the serovars Pomona and Icterohaemorrhagiae, in hamsters was determined as 106.893 organisms and 107.38 organisms, respectively and the challenge was carried out with 100 LD50 (≈109 ) organisms, on 28th day post first immunization. Challenge studies revealed maximum protection levels of 80 to 100 per cent in groups immunised by modified inactivated vaccine alone and combination of rLigA and inactivated vaccine. Groups immunised with rLigA protein alone showed 60-70 per cent protection to both serovars. The highest MAT titres to homologous and heterologous serovars were presented by the groups immunized with a combination of rLigA protein and modified inactivated vaccine. These groups elicited higher MAT titres to heterologous serovar Icterohaemorrhagiae compared to whole cell vaccine alone, which indicated the genus specificity contributed by the partial rLigA protein. It also showed that the inactivated vaccine and recombinant protein compliment each other in increasing the respective immunogenicity. The study revealed that IFA adjuvanted rLigA protein could elicit the maximum ELISA titres in hamsters, followed by the group immunised by IFA adjuvanted rLigA protein combined with modified inactivated vaccine. The IFA adjuvanted vaccine groups showed higher ELISA titres compared to those adjuvanted with aluminium hydroxide but the aluminium hydroxide adjuvanted vaccine groups showed consistent increase in antibody titres.ThesisItem Open Access DETECTION AND MOLECULAR CHARACTERIZATION OF BENZIMIDAZOLE RESISTANCE IN GASTROINTESTINAL NEMATODES OF GOATS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-06-23) ASHA RAJAGOPAL; Lucy SabuThe study was conducted with the objectives of assessing the status of benzimidazole resistance in gastrointestinal (GI) nematodes of goats in Kerala, detection of single nucleotide polymorphisms (SNPs) associated with benzimidazole resistance in the β-tubulin gene of predominant GI nematode species and to evaluate the efficacy of the egg hatch assay, larval development assay and PCR-RFLP in detection of benzimidazole resistance. Microscopical examination of 520 faecal samples collected from goats from 10 organized farms and 16 small holder farmers’ flocks in eight agro-ecological zones of Kerala revealed an overall prevalence of 81.5 + 5.54 per cent strongyles in goats. There was significant difference between the prevalence of strongylosis in organized farms (91.19 + 4.33 %) and small holder farmers’ flocks (65.34 + 10.27 %). The mean faecal egg counts (FECs) also differed significantly between organized farms and small holder farmers’ flocks. The prevalent genera of strongyles identified on coproculture were Haemonchus spp., Trichostrongylus spp. and Oesophagostomum spp. Faecal egg count reduction test (FECRT) done for screening the benzimidazole resistance status in 10 organized farms and 16 small holder farmers’ flocks identified benzimidazole resistance in all the organized farms. Among the small holder farmers’ flocks, 43.75 per cent were found to be resistant to benzimidazoles. Susceptibility was identified in 37.5 per cent of the small holder farmers’ flocks while resistance was suspected in 18.75 per cent of the flocks. Statistical analysis revealed significant association between the resistance status and farm type. Haemonchus spp. was found to be the most predominant GI nematode in post-treatment faecal cultures indicating that it is the major species responsible for benzimidazole resistance. Resistance status was found to be significantly correlated with the frequency of deworming in flocks. Molecular genotyping by PCR-RFLP revealed E198A polymorphism in isotype 1 β-tubulin gene in Haemonchus spp. with an overall frequency of 0.516 for the resistant allele (r). The overall prevalence of homozygous resistant genotype (rr) at codon 198 in Haemonchus spp. was 25.6 per cent. No polymorphism was identified at codons 167 and 200 in Haemonchus spp. in this study. In Trichostrongylus spp., F200Y polymorphism was identified in isotype 1 β-tubulin gene with an overall gene frequency of 0.337 for the resistant allele and with 28 per cent of the larvae genotyped being homozygous resistant (rr). Susceptible genotype was identified at codons 167 and 198. All the Oesophagostomum spp. larvae genotyped were found to be of the susceptible genotype at codons 198 and 200. In vitro detection of benzimidazole resistance was done by egg hatch assay (EHA) and larval development assay (LDA). Correlation of the results of FECRT, EHA, LDA and PCR-RFLP revealed significant correlation (p < 0.05) between FECR per cent, ED50 in EHA, Pdd (proportion of larvae hatching at the discriminating dose of 0.02 µg/ml) in LDA and the percentage of homozygous resistant (rr) genotype in PCR-RFLP. There was significant correlation between ED50 and Hdd (hatching ratio of strongyle eggs at the discriminating dose of 0.1 µg/ml) values in egg hatch assay. Pdd values were found to be significantly correlated with other resistance parameters indicating that it is a better criterion for resistance detection than LD50 in LDA. To predict the genotypic resistance using phenotypic resistance indicators, regression equations were derived with rr genotype per cent as dependent variable and FECR per cent, ED50 and Pdd as the independent variables.ThesisItem Open Access CONSERVATION ECOLOGY BASED ON PARASITE-NUTRIENT-SEASONAL INTERACTIONS IN NILGIRI TAHR (Nilgiritragus hylocrius)(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-12-30) BIJU S.; P. C. SaseendranThe study was conducted to evolve suitable strategies for the conservation of the endangered Nilgiri tahr (Nilgiritragus hylocrius Ogilby, 1838) by investigating the primary interaction of the species with its parasites, forages and the season. The three habitats of Eravikulam, Nelliampathi and Kochupamba were selected and the study was conducted in the three seasons of summer, monsoon and post-monsoon. The indicators selected were incidence of parasitism, the degree of parasitic infection, the nutrient composition of forage, health status of the herd, stress level of animals, habitat features, herd structure and the general behavior of the herd. The indices in this regard were examined and recorded across the seasons. The herd size recorded ranged from 12 in Kochupamba to 36 in Eravikulam with adult females comprising nearly half of the herd. The habitats were typically of grass lands with short grasses adjacent to rocky cliffs. The activity budget was recorded to assess the behavior of the animals and it revealed grazing (37.4%) and resting (23.3%) as the prominent activity in tahr with very low share of browsing (0.8%) in the species. The activity budget also observed minor shifts between seasons, habitats and in age-sex class. The predominant grass species in each habitat were identified and the nutrient composition of the grasses was analysed showing significant difference between seasons in dry matter, crude protein, crude fibre, ether extract, total ash and nitrogen free extract. The body condition score (BCS) of all the animals ranged from three to five indicating the herds to be healthy across all seasons. There were no reports of disease or mortality in the animals in the study period. A total parasitic incidence of 38.9 per cent was observed and the degree of infection was assessed to be mild as indicated by the eggs per gram (EPG) below 1000 in the observed data. Strongyles and coccidia were the major parasites with 34.3 per cent and 15.5 per cent incidence respectively and the other parasites recorded were Moniezia, Trichuris, Capillaria, and Strongyloides. There was significant difference in parasitic incidence between habitats and seasons, with higher share in monsoon (58.9%) followed by summer (40%) and post-monsoon (17.8%). Eggs per gram also observed significant seasonal difference with higher count in monsoon (360.64) followed by post-monsoon (232.81) and summer (114.29). The faecal cortisol metabolite level estimated by enzyme immunoassay was significantly different between habitats, with Eravikulam recording a higher mean value of 472.17 ng/g, and the difference was non-significant between seasons. Significantly higher faecal cortisol metabolite was observed in parasite infected animals compared to non-infected animals. The study identified the significant presence of parasites in tahr population, indicating the dynamism and balance in equilibrium of host-parasite relationship which is a major driver in the organization of the ecosystem. The energy flow dynamics across seasons and habitats significantly influence the interrelationship of the host and parasite.ThesisItem Open Access YIELD AND QUALITY OF TRANSVAGINALLY RETRIEVED OOCYTES IN NORMAL AND REPEAT BREEDING CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-12-01) ABHILASH R. S; ABHILASH R. S; Metilda Joseph; Metilda JosephThe study was conducted to assess the efficacy of transvaginal oocyte recovery (TVOR) and to evaluate the quality of oocytes recovered from 12 normal and 12 repeat breeder crossbred cows stationed at the University Livestock Farm and Fodder Research Station, Mannuthy. Both normal and repeat breeder cows further divided into two groups of six animals each based on the TVOR frequency. Transvaginal oocyte recovery was performed at a frequency of once weekly in groups I and III and twice weekly in group II and IV for a period of two months (Group I and II- normal cows, and group III and IV- repeat breeders, six animals each). Frequency of TVOR or reproductive status of animal was not found to influence ovarian biometry. Number of small ( 9 mm) follicles were significantly lower (p ≤ 0.05) in normal and repeat breeder cows subjected to twice weekly TVOR. The number of follicles aspirated, number of oocytes retrieved, culture grade oocytes, matured oocytes, fertilized oocytes, cleaved oocytes per session and oocyte recovery rate (%) were siginicantly higher (p ≤ 0.05) in normal animals and those animals subjected to twice weekly TVOR. However, there was no significant difference between maturation rate, fertilization rate and cleavage rates between normal and repeat breeder animals and animals subjected to once and twice weekly TVOR. Evaluation of oocyte maturation rate using Hoechst 33342 and FDA also revealed that maturation rate was higher in normal breeders. No significant difference in the serum progesterone and blood urea nitrogen level were observed when normal and repeat breeder cows were subjected to TVOR at varying frequencies. A significantly higher follicular fluid progesterone concentration was observed in normal animals when compared to repeat breeders. The present study revealed that oocyte quality in repeat breeders is significantly lower than normal breeders, and TVOR at twice weekly interval was found to be an effective tool for harvesting maximum number of oocytes from crossbred cows.ThesisItem Open Access IDENTIFICATION, ISOLATION AND CHARACTERIZATION OF POTENTIAL ACARICIDAL MOLECULE(S) FROM PLANT EXTRACT AND STUDIES ON THEIR MODE OF ACTION(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2015-09-30) SREELEKHA K. P.; Sanis JulietThe present study was conducted to isolate active biomolecule(s) from Blumea mollis plant extract having potent acaricidal activity against R. (B.) annulatus and to study their mode of action. Blumea mollis (D. Don) Merr., Asteraceae family is an annual aromatic herb indigenous to tropical South India. The plant materials were collected from identified phytogeographical zones. The ethanolic extract of different accessions of the plant was assayed for acaricidal activity using adult immersion test and larval packet test. Further, isolation and characterization of bioactive fractions / sub-fractions were performed using standard chromatographic and spectral techniques. The accession CAS 01 of B. mollis was selected as the best accessions with more than 80-100% efficacy against R.(B.) annulatus.The freeze dried crude extract of elite accession of CAS-01 was tested for safety using OECD guidelines. Phytochemical analysis revealed the presence of high content of terpenoids in the accession CAS 01. The hexane fraction was found to be active and HPTLC profiling showed marker concentration comparable to that of ethanolic extract of CAS 01. Phytochemical analysis of hexane fraction revealed the presence of high content terpenoids further confirmed by GC-MS. The GC-MS analysis of active sub-fractions revealed the presence of several components ranging from monoterpenes, diterpenes to sesquiterpenoids. Among the four molecules selected based on their chemical nature and area percent, ethyl palmitate, VP5 and marker compound VP1 elicited the acaricidal activity varying from 95 - 100 %. The histology and transmission electron microscopic analysis of active biomolecules exhibited marked changes comparable to that of synthetic acaricides deltamethrin and amitraz. The β octopamine receptor gene expression studies using whole tick and ovary showed significant down regulation comparable to amitraz in case of VP1 whereas ethyl palmitate and VP5 showed no significant change. Structural analogy results revealed similarity with compounds mediating action through GPCR receptors. The isolated molecules can be used as template for the development of promising acaricides.ThesisItem Open Access BEHAVIOURAL, PHYSIOLOGICAL AND BIOCHEMICAL STRESS RESPONSES OF CROSSBRED COWS TO VARYING THERMAL INDICES IN DIFFERENT MANAGEMENT SYSTEMS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-06-23) HARIKUMAR S.; Anil K. S.1. Study the behavioural, physiological and biochemical responses of crossbred cows to thermal stress. 2. Assessment of different management systems for alleviating thermal stress of crossbred dairy cowsThesisItem Open Access CHARACTERIZATION OF MALABARI BUCK SEMINAL PLASMA PROTEINS IN RELATION TO SEMEN FREEZABILITY AND FERTILITY(COLLEGE OF VETERINARY AND ANIMAL SCIENCESMANNUTHY, THRISSUR, 2016-12-30) AMBILI JOHN; Hiron M. HarshanThe study was undertaken to characterize the protein profile of Malabari buck seminal plasma for identifying protein markers of freezability and fertility and to compare the in vivo fertility of Malabari buck semen with low and high semen freezability. Adult Malabari bucks with post thaw sperm progressive motility of more than 35 per cent were classified as having high semen freezability while those with less than 30 per cent were classified as having low semen freezability (four each) were utilized for the study. Tris - egg yolk based extender was used for cryopreservation of the ejaculates (n = 48; 24 each from bucks having high and low semen freezability). Significantly higher values were noted in the pre-freeze acrosome integrity and membrane cholesterol levels and post-thaw motility, viability, acrosome integrity, hypo osmotic sperm swelling response (HOS) and membrane cholesterol levels of high semen freezability bucks when compared with low semen freezability bucks. Whereas the seminal plasma ALP and LDH were found to be significantly lower in high semen freezability bucks compared with low semen freezability bucks at post thaw stage. The total protein content and average number of protein bands in seminal plasma of high and low semen freezability bucks did not differ significantly (75.82 ± 4.62 mg/ml vs 67.07 ± 6.07 mg/ml; 20.83 ± 0.53 vs 22.09 ± 0.49). The 24 kDa, 79 kDa, 84 kDa and 134 kDa proteins were found to have significantly higher occurence in seminal plasma of bucks with high semen freezability whereas the occurence of 15 kDa, 19.9 kDa, 21.5 kDa, 70kDa and 89 kDa proteins were found significantly higher in seminal plasma of bucks with low semen freezability. On analysis of 2D gels, an average of 67.67 ± 4.06 and 75.33 ± 4.28 spots (pI 3-10) were detected in semen of high and low semen freezability bucks, respectively and the difference was not significant. On western blot analysis, osteopontin, a protein fertility marker, was found to be present in seminal plasma of all the bucks studied. For fertility studies 134 does were inseminated with semen preserved by chilling and 91 with cryopreserved semen. The conception rate (CR) with cryopreserved semen was significantly higher in bucks with high semen freezability than with low semen freezability. In the semen preserved by chilling, no significant difference was noticed between low and high semen freezability animals in the spermatozoa progressive motility, viability, acrosome integrity, HOS response or seminal plasma ALP and LDH levels after dilution or after 24 h of storage at refrigeration. But when chilled semen was used, the CR was significantly higher for bucks with low semen freezability than for bucks with high semen freezability.
