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ThesisItem Open Access CHARACTERIZATION OF Clostridium perfringens MEMBRANE VESICLES AND THEIR IMMUNOGENIC POTENTIAL IN MICE(Assam Agricultural University, Khanapara,Guwahati, 2016-07) DEURI, NIRAB KUMAR; SHARMA, R. K.The present study was undertaken with a view to characterize as well as evaluate the immuno-protective potential of the Membrane Vesicles (MVs) of Clostridium perfringens Type ‘A’. A total of five isolates of C. perfringens from the repository of Department of Microbiology, College of Veterinary Science, Khanapara, Guwahati were revived and reconfirmed based on gross morphology, staining characteristics and amplification of 16S rRNA and cpa genes by polymerase chain reaction. One isolate of C. perfringens belonging to Type A was selected and grown in TPGB and RCMB media. MVs were extracted at 4, 8, 12 and 24 hrs of growth. Study of protein profile based on SDS-PAGE revealed the appearance of one band at 4 and 8 hrs of growth and 7 bands in MVs extracted at 12 hrs of growth on TPGB. No bands were observed in MVs extracted at 24 hrs of growth in TPGB. Molecular weight of the protein bands were ranging from 43.3 kDa to 75.2 kDa. The MVs extracted from RCMB media revealed the appearance of one protein band in 4 and 8 hrs and 6 bands from 12 hrs of culture growth. The 24 hrs growth culture in RCMB also revealed no bands in SDS-PAGE. The RCMB protein bands were also ranging from 43.3 kDa to 75.2 kDa. The extracellular proteins and cell lysate proteins were extracted from the cultures grown on BHI broth and compared the protein profile of MVs as revealed by SDS-PAGE. The protein profile revealed 15 distinct bands in cell lysate proteins 11 bands in MVs and 4 bands in extracellular proteins. Cell lysate protein bands were ranging from 17.2 kDa to 75.0k Da, whereas MVs and extracellular protein bands were ranging from 43.3 kDa to 75.0k Da and 44.1 kDa to 75.0 kDa, respectively. The DNA content of MVs was released by GES reagent and PCR was done targeting to 16S rRNA and cpa (alpha toxin) genes yielding two distinct bands of 795 bp and 324 bp. Immunization of mice to group A1 and B1 was done with the vaccine prepared from MVs at the dose rate of 20 µg/0.2 ml while group A2 and B2 was done with that of 40 µg/0.2 ml through intraperitoneal (i/p) route at 0, 14, and 28 days. The protective efficacy of the MVs vaccine was studied by challenging the immunized mice on 42nd day post-primary vaccination with 3x108 CFU and 6x108 CFU of C. perfringens Type A. The challenge trial revealed that the 20.0 µg and 40.0 µg /0.2ml dose of vaccines could confer 100.0 percent protection in the immunized mice following homologous challenge with 3 x 108 CFU. However, the challenge trial with 6 x 108 CFU of homologous strain of C. perfringens Type A revealed only 66.66 and 33.33 percent protection in the mice immunized with 40.0 µg and 20.0 µg /0.2 ml vaccine doses, respectively.ThesisItem Open Access EFFECT OF ANTIOXIDANTS ON IN-VITRO MATURATION OF VITRIFIED BOVINE OOCYTES(Assam Agricultural University, Khanapara, Guwahati, 2016-07) SONOWAL, JOYSHIKH; Barua, P. M.A total 717 ‘A+B’ grade oocytes were collected by aspiration cum slicing techniques from 294 numbers of cattle ovaries obtains from abattoir within 1-2 hours of slaughter. A total 651 ‘A+B’ grade immature oocytes were vitrified by two-step vitrification technique with combination of ethylene glycol and glycerol @ 7M concentration. Five hundred twenty eight numbers of normal vitrified-thawed bovine follicular oocytes were classified in to 8 experimental groups consisting of 66 numbers of oocytes in each and conducted in-vitro maturation as vitrified control (without antioxidant) and supplemented with different concentration of Vitamin E (50µM, 100µM, 200µM) and vitamin C (50µM, 100µM, 200µM) and combination of vitamin E (100µM) + vitamin C (100µM) groups. Non-vitrified 66 numbers of oocytes were used for in-vitro maturation in media without supplementation that served as control media. The rate of cumulus cells expansion and polar body formation was significantly (P<0.01) higher in non-vitrified control (85.18 ±2.57 and 65.38±1.83, respectively)group than vitrified (51.67±1.94 and 30.26±0.16, respectively) control group of bovine follicular oocytes. The effect of addition of antioxidants viz. vitamin E and vitamin C in media at different concentrations (50µM, 100µM, 200µM) were studied separately. The rates of vitrified bovine follicular oocytes with cumulus cells expansion and polar body formation were 56.39±3.49, 69.95±3.20 and 54.44±4.73 per cent and 34.62±1.83, 56.23±1.61 and 31.62±4.50 per cent in medium supplemented with 50µM, 100µM and 200µM of vitamin E, respectively and 52.79±1.39, 71.19±2.63 and 51.17±3.59, per cent and 33.56±2.18, 54.57±1.69 and 30.43±2.01, per cent in medium supplemented with 50µM, 100µM and 200µM of vitamin C, respectively. The mean percentage of cumulus cells expansion and polar body formation was significantly (P<0.01) higher in media supplemented with 100µM of vitamin E or vitamin C group than 50µM and 200µM of vitamin E or vitamin C groups and vitrified (without antioxidant) control group of oocytes. The rate of cumulus cells expansion and polar body formation of vitrified bovine follicular oocytes was significantly (P<0.01) higher in group supplemented with combination of vitamin E + vitamin C @ 100µM of each (83.23±3.00 and 61.25±3.38, respectively) than vitrified control (51.67±1.94 and 30.26±0.16, respectively) group. While comparing the best concentration groups of all experimental groups, DMRT indicated that the mean percentage of cumulus cells expansion and polar body formation was significantly (P<0.01) higher in combination of vitamin E @100 µM + vitamin C @100 µM group and non-vitrified (control) group than vitamin E @100 µM, vitamin C @100 µM groups. Vitrified control group was significantly (P<0.01) lower than the other experimental groups. The result of the present study showed that the rate of vitrified bovine follicular oocytes in respect of cumulus cells expansion and polar body formation was significantly (P<0.01) higher when antioxidants like vitamin E @ 100 µM or vitamin C @ 100 µM were added as compared to vitrified (without antioxidant) control. Each of vitamin E (100 µM) and vitamin C (100 µM) supplemented in TCM-199 improved the rate of in-vitro maturation of vitrified bovine follicular oocytes. Vitamin E@ 100µM + vitamin C@ 100µM supplemented in TCM-199 showed the highest rate of in-vitro maturation of vitrified bovine follicular oocytes.ThesisItem Open Access MOLECULAR EVALUATION OF BULL SEMEN IN RELATION TO CERTAIN FERTILITY MARKERS(Assam Agricultural University, Khanapara, Guwahati, 2016-07) BORGOHAIN, INDRANI; DUTTA, D. J.The present study was conducted for molecular evaluation of bull semen and to study its relationship with different semen characteristics. Fresh semen samples were collected from six breeding bulls. A total of six ejaculates were collected from each of six healthy breeding Jersey bulls at 4 days interval. Immediately after collection, the samples were subjected to physio-morphological and biomolecular evaluation. Percentage of Hypo-osmotic Swelling Test (HOST) positive spermatozoa and acrosome-intact sperms increased with an increase in initial sperm motility. Biochemical evaluation indicated that seminal plasma protein, sperm protein and sperm cholesterol concentration increased with higher motility. The protein profiling revealed the highest band intensity of a 25 kDa protein in both seminal plasma and sperm membrane of all the bulls. Whereas, proteins of 104-92 kDa molecular mass were absent in the bulls with initial sperm motility below 80 per cent in both seminal plasma and sperm membrane. Expression of Chaperonin Containing T-complex protein 1, subunit 8 (CCT8) gene was found to be negatively correlated with sperm motility, whereas the expression of Adenylate Kinase 1 (AK1) gene did not show any significant relationship with sperm motility.