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ThesisItem Open Access CHARACTERISATION OF GENES AFFECTING METABOLIC ADAPTATION IN TRANSITION DAIRY COWS(College of Veterinary and animal Science,Mannuthy, 2019) VASUDHAR BHAT S.V.; Shynu M.The period from three weeks before parturition until three weeks after parturition is the transition period in dairy cows. This time period is critical in the health and production of dairy cows and profitability of dairying as most diseases occur during this period. The dairy cows undergo several metabolic adaptations to overcome the stress of foetal growth and lactation. Even when maintained under similar feeding and managemental conditions, dairy cows exhibit variation in adaptability. This indicates the genetic influence on metabolic adaptability. The present study was performed in 30 apparently healthy pregnant crossbred dairy cows with an objective to characterize three genes responsible for metabolic adaptability and to detect variations in these genes, among animals with varying adaptability, as indicated by their BHBA concentration. Blood was collected and serum was separated at fortnightly intervals from four weeks before the predicted day of parturition until four weeks after parturition. Concentration of NEFA and BHBA were determined. The mean concentration of NEFA (0.254±0.21 mmol/L) and BHBA (0.608±0.024 mmol/L) during pregnancy were significantly elevated in comparison with the NEFA (0.349±0.37 mmol/L) and BHBA (0.681±0.23 mmol/L) during lactation. As BHBA is a better indicator of metabolic adaptability, the animals were grouped based on their BHBA concentration using cluster analysis. The genes DNAJC30, SNAI2 and UEVLD known to influence metabolic adaptability were sequenced from representative animals of each group. Four differences (transitions) in sequence were observed downstream the coding region of DNAJC30 gene between the groups. One change was located in the promoter region and another in exon one of SNAI2 gene. The CDS of UEVLD gene did not show any change when compared with the available Bos taurus sequence in NCBI. The variations in genes studied might contribute to difference in the metabolic adaptability between the groups. Further studies in a larger population, is required to ascertain the suitability of using these as markers for selection of dairy animals for better metabolic adaptabilityThesisItem Open Access ASSESSMENT OF IMMUNOMODULATORY EFFECTS OF GOAT LACTOFERRIN AND ITS PEPSIN HYDROLYSATE(College of Veterinary and animal Science,Mannuthy, 2019) DEEPAK CHANDRAN; UMA R.Lactoferrin, well known as a minor whey protein, is an 80kDa iron-binding glycoprotein primarily present in milk. It exhibits an array of biological activities including antioxidant, antimicrobial, anticancer, metal binding and immunomodulatory properties. Many studies point out that whey proteins contain various bioactive peptides in their primary sequences whose immunoregulatory and therapeutic potentials have not been much explored. The present study focussed on the isolation and characterisation of lactoferrin (gLf) from the colostrum/milk of Malabari goats, followed by the preparation of its pepsin hydrolysate (gLPH) so as to assess their in vitro immunomodulatory potentials on peripheral blood mononuclear cells (PBMCs). Colostrum samples collected from Malabari goats maintained at University Goat and Sheep Farm, College of Veterinary and Animal Sciences, Mannuthy were processed and treated with ammonium sulphate to remove globulins from the samples. Fractions containing albumin and remaining proteins including lactoferrin after dialysis were loaded on to CM- Sephadex C-50 cation exchanger column and eluted with a step gradient of 0.4, 0.6 and 0.8M NaCl. The presence of gLf in the high OD280 value fractions eluted with 0.8M NaCl was confirmed by 12 per cent SDS-PAGE and Western blotting. The concentration of gLf as estimated by Lowry’s method was found to be 15.103 mg/L of colostrum. The gLf was hydrolysed by treatment with three per cent porcine pepsin under acidic conditions at 37 °C for four hours to form gLPH. The immunomodulatory potentials of gLf and gLPH were studied on bovine PBMCs which were cultured along with specific combinations of mitogen, phytohaemagglutinin (PHA)/gLf/gLPH at 37°C for 72h in five per cent CO2 humidified air. A wide range of concentrations of both gLf and gLPH were utilized to assess their cytoproliferative effect on PBMCs with or without mitogens. In the presence and absence of mitogen, higher concentrations of lactoferrin were found to significantly inhibit the proliferation of PBMCs whereas lower concentrations brought about significantly active proliferation of the cells. Maximum proliferation with or without mitogen was observed with 1.5 µg/mL of culture. The cell proliferation potentials of gLPH was higher than that of gLf. With gLPH, in the presence and absence of mitogen, maximum proliferation of PBMCs could be detected with the highest concentration of 50 µg/mL. The cells cultured with gLf and gLPH showing maximum cell proliferation were harvested and their RNA were isolated to study the expression of pro-inflammatory cytokines by real time PCR. It was demonstrated that gLf and gLPH showed potentiated anti-inflammatory activity by significantly inhibiting the expression of the pro-inflammatory cytokine genes IL1β, IL-6 and TNF-α.ThesisItem Open Access MOLECULAR SEXING OF GREEN-CHEEKED CONURE (PYRRHURA MOLINAE) USING CHD1 AND NIPBL GENES(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2014) PRASHANT UDAGATTI; Shynu M.Sexing in birds is important for behavioral and ecological studies and also to improveex situ captive breeding programme. More than 50% of the world’s avian population are monomorphic and are difficult to sex phenotypically. Some avian species exhibit marked sexual dimorphism as adults, but lack dimorphism as juveniles. Monomorphic nature of many pet birds creates problems for pet breeders in identifying the sex. Traditional methods of avian sexing were based on behavioral observations, difference in size and shape, differences in the sounds produced, cloacal examination, surgical techniques and cytogenetic analysis. Most of them are time consuming, invasive and often generate ambiguous results. In the present study, PCR based techniques were used to identify the sex of a popular pet bird, Pyrrhura molinae (Green-cheeked conure), which is monomorphic in nature. The gene CHD1 (encoding chromo helicase- DNA-binding protein 1) is located in both the sex chromosomes, Z and W of birds.Intron 9 of CHDI gene was amplified by PCR technique in DNA isolated from the feather. Retroposon insertion in the region of amplification in Z chromosome generated a larger fragment. As female birds are heterogametic (ZW) PCR amplification generated fragments of two different sizes- 1100 bp and 600 bp, whereas in male birds (ZZ), only a single fragment of 1100 bp was observed. It revealed two different patterns in male and female birds which help to identify the sex accurately. Intron 16 region of CHDI gene was also amplified to see for sex specific difference in amplicon pattern between male and female birds. Though five out of thirty birds generated distinct amplification pattern rest of the birds did not generate unambiguous results. The result was similar for NIPBL intron 16 also. It appears that amplification of CHD1 intron 9 region is the most reliable method in sexing of green cheeked conure. As the method is non-invasive, it produces minimum stress to the bird and can be employed for easy and accurate sexing of the bird.ThesisItem Open Access METABOLIC PROFILE OF CROSSBRED DAIRY COWS DURING TRANSITION PERIOD(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2017) ANISHA J. PERUMBILLY; Shynu M.Dairy cows are considered to be most prone to diseases during the period from late pregnancy to onset of lactation, i.e. during the transition period. The present investigation was carried out in blood/serum collected at fortnightly intervals from 15 crossbred dairy cows, from eight weeks before the predicted date of calving till eight weeks after calving, with the objectives of generating a metabolic profile and for evaluating the antioxidant status. Concentrations of glucose, NEFA, BHB, cholesterol, urea, albumin, ceruloplasmin and haptoglobin were determined; differential leukocyte count was done; and assessment of oxidative stress was also performed. The mean concentration of glucose (47.35±1.32 mg/dL) and cholesterol (95.83± 3.62 mg/dL) during transition period was significantly lower than pre and post-transition period. NEFA (0.576 ± 0.08 mmol/L) and BHB (0.638 ± 0.05 mmol/L) concentrations reported significant increase during transition when compared to pre-transition period. Concentration of indicators of protein status viz. albumin and urea were 3.39 ± 0.09 g/dL and 12.26 ± 0.66 mg/dL respectively during transition period and did not differ significantly from pre or post-transition period. Out of the two acute phase proteins measured, ceruloplasmin did not show significant variation during the study period, but a significant increase was shown by haptoglobin during transition period. The level of MDA, an indicator of oxidative stress was higher during transition period, indicating significant oxidative stress during the period. TAS did not show significant change but the antioxidant status of the animals could not be considered optimum, as eleven out of fifteen animals exhibited diseases albeit transient during the period. A significant increase in the number of neutrophils and monocytes and a highly significant decrease in the number of lymphocytes observed during transition period could be due to the influence of corticosteroids. A comprehensive study involving more number of transition animals, maintained under different managemental conditions shall help in establishing reference intervals for various analytes during period.ThesisItem Open Access MOLECULAR FINGERPRINTING OF Riemerella anatipestifer ISOLATES FROM DUCKS OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2012) SHONIMA P.; Uma RThe present work is aimed at exploring the genetic diversity of R. anatipestifer isolates from ducks of Kerala by molecular fingerprinting method. Isolation of R. anatipestifer was attempted from 163 samples and of which six isolates were obtained, namely KAL-1, KNB-1, KML-1, KML-2, KML-3, and KTL1. The isolates were non-haemolytic on sheep blood agar, did not grow on MacConkey’s agar, catalase and oxidase positive, indole negative, ornithine decarboxylase negative and gelatin liquefaction positive. All the isolates were subjected to specific 16S rRNA based PCR and all produced a 665 bp fragment. Sequencing the fragment revealed 99% similarity to R. anatipestifer strain ATCC 11845 ribosomal RNA partial sequence (Accession no. NR 026025.1). Riemerella anatipestifer species-specific PCR yielded the expected 546 bp fragment. Molecular fingerprinting was done in the six isolates using ERIC-PCR which produced 10-12 bands per isolate ranging in size from 150 bp to 2000 bp. A 700 bp fragment was common to all the six isolates. Software analysis of ERIC-PCR revealed four variants namely E1 (KML-1 and KML-2), E2 (KNB-1 and KML-3), E3 (KAL-1 and E2), and E4 (KTL-1). This showed that, isolates originating from the same flocks were genetically identical, while those from different regions of Kerala had different fingerprints. Our results suggest that the R. anatipestifer of various genetic make-up prevails among ducks of Kerala and the information would be helpful in developing proper control and therapeutic strategy against the emerging New Duck disease in Kerala.ThesisItem Open Access MOLECULAR STUDIES ON TAPASIN GENE IN CHICKEN, DUCK AND GUINEA FOWL(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) VARUNA P. PANICKER; Uma RThe present study was undertaken to investigate the genetic make up of exon 5 – exon 6 region of avian tapasin gene in White Leghorn, White Plymouth Rock, Rhode Island Red, Kadaknath and Naked neck breeds of domestic fowl, Kuttanad and White Pekin breeds of duck and guinea fowl. The genomic DNA extracted from the blood of each breed was amplified using primers specific to exon 5 – exon 6 of chicken tapasin. All the amplified products were of 400 -600 bp size. The products were extracted from gel and were cloned in PTZ57R/T vector. The cloned products were then sequenced by automated method. The tapasin exon 5 – exon 6 sequence data of the various breeds studied varied both between species and between breeds in their length. The size of amplicon was 582 bp for all the chicken breeds except Naked neck which was found to be of 552 bp size. The PCR product size of the duck breeds Kuttanad and White Pekin were 440 bp and 445 bp respectively; and that of guinea fowl was 582 bp. All the obtained nucleotide sequences were then analyzed by comparing with tapasin exon 5 – exon 6 sequence of chicken obtained from the database GenBank. The results showed that the GenBank sequence had maximum homology with the chicken breeds studied and, followed by guinea fowl and the least percent similarity was with the duck breeds. Among the exotic chicken breeds White Leghorn was 100 % identical to the database sequence where as White Plymouth Rock exhibited 99.1 % and Rhode Island Red showed 98.8 % homology to the published sequence. The native breeds Kadaknath and Naked neck were found to be 99 % and 98.2 % homologous respectively, to GenBank sequence. RE profile analysis of the sequence data showed that guinea fowl and all the chicken breeds were having almost similar restriction pattern for Sma I and Pvu II. Some chicken breeds had cut sites for Alu I. Duck breeds separated themselves out in that their DNA could be cut only by Alu I. Comparison of deduced amino acid sequences revealed variations between the different species studied. White Leghorn had the same amino acid sequences as those of AJ004999. Rhode Island Red varied from the GenBank sequence at three positions. All the other fowl breeds possessed four amino acid replacements and guinea fowl exhibited changes at nine positions when compared to the data base sequence. In the case of duck breeds, amino acid sequences obtained varied much from other species as well as in between the breeds due to variation in their nucleotide sequences. Phylogenetic analysis of the nucleotide sequences and the deduced amino acid sequences of different species and breeds revealed clustering of different chicken breeds along with guinea fowl as one main branch. Duck breeds clustered to a separate branch of the phylogenetic tree. The secondary structure of tapasin exon5 - exon6 region of various chicken breeds, duck and guinea fowl when predicted using the PAPIA system made it clear that the structures were similar in chicken breeds and guinea fowl reflecting their high degree of amino acid sequence identity while White Pekin and Kuttanad duck breeds showed variations in structure as expected owing to their amino acid sequence dissimilarities with respect to the other two species.