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2010 - 20175
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Subject: Veterinary Physiology × End date: 2019 × Start date: 2010 × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    LEPTIN RECEPTOR EXPRESSION IN CULTURED OVARIAN FOLLICLES OF SHEEP
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-05) SRAVANI PRAGNA, K; Siva Kumar, A.V.N(MAJOR); Rambabu Naik, B; Punya Kumari, B
    ABSTRACT : The expression of Leptin receptor (LepR) mRNA and protein was studied in sheep using Real time quantitative PCR and Immunohistochemistry in the cumulus cells and oocytes from : (i) In vivo grown preantral, early antral, antral, large antral follicles and Cumulus Oocyte Complexes (COCs) obtained from large antral follicles and subjected to 24h of In Vitro Maturation ( IVM) and (ii) Preantral follicles (PFs’) exposed to TCM199B, TCM199BL (10ng/mL) and TCM199BHGL (10ng/mL) media for 3min, cultured in vitro for two, four or six days and subsequently matured in vitro for 24h in respective cultures separately. LepR was observed at all stages of in vivo grown and in vitro cultured ovarian follicles in both cumulus cells and oocytes. Expression levels and patterns of LepR mRNA in PFs’ cultured in TCM199BHGL was similar to in vivo in all the stages except in cumulus cells from COCs after in vitro maturation for 24h, where the expression was significantly higher which was a positive effect. The expression of LepR in cumulus cells among the three different in vitro culturing conditions was significantly higher in PFs’ cultured in TCM199BHGL medium. In the oocytes, the Leptin receptor expression was similar or significantly higher in PFs’ cultured in TCM199BHGL medium than in vivo grown follicles except in PFs’ exposed to 3min stage which could suggest the synergistic action of growth factors and hormones with Leptin. Immunohistochemistry studies revealed highest intensity of Leptin receptor protein expression in the oocytes. Mild to moderate staining was observed in cumulus cells with good intensity in large antral stages. This protein expression coincided with gene expression. From our studies it is concluded that Leptin along with other growth factors and hormones supplementation stimulated the expression of Leptin receptor mRNA and protein on par with that of in vivo /even higher than that of in vivo condition.
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    ThesisItemOpen Access
    INFLUENCE OF LEPTIN ON THE EXPRESSION OF p53 and BAK GENES IN CULTURED OVARIAN FOLLICLES IN SHEEP
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2015-11) PRABHAVATHI, T; RAO, V.H(MAJOR); SIVA KUMAR, A.V.N; VEERABRAHMAIAH, K
    ABSTRACT : The present study was conducted to know the influence of Leptin on the expression of p53 and BAK (apoptotic) genes in the sheep preantral follicles (PFs’) cultured in vitro. Ovaries of sheep were collected from the local slaughter house. Intact PFs’ (250-400μm) were isolated and placed individually in 20μl droplets of standard medium (TCM 199 B + 2 μg/ml FSH, 0.1μg/ml T4 + 10 ng/ml IGF-I + 1 mIU/ml of GH) supplemented with Leptin (10ng/ml) for 2, 4 or 6 days. Quantitative expression of Test genes (p53 and BAK) and Reference genes (18s RNA, RPLPO, HPRT1) were studied in the cumulus cells and oocytes isolated at different developmental stages (preantral, early antral, antral and large antral follicles) of the in vivo and in vitro grown follicles. The entire experiment was repeated twice. Duplicate samples of cDNA from each replicate of the experiment were subjected to relative-RT-qPCR. The pattern of expression of p53 gene in the cumulus cells from ovarian follicles grown in vivo and cultured in Leptin was similar from preantral to large antral follicle stage. In the oocytes, however, such similarity was restricted up to the antral follicle stage only. It is concluded that Leptin in culture medium mimiced the pattern of p53 expression in in vitro as in in vivo to some extent. The BAK gene expression was undetected in the cumulus cells and oocytes isolated from all the stages of both in vivo and in vitro developed ovarian follicles. Accordingly it is concluded that (i) Leptin has no influence on the expression of BAK gene in cultured ovarian follicles in sheep (ii) BAK may not be an important regulator of apoptosis in ovarian follicles and (iii) p53 plays a relatively more significant role than BAK in the regulation of apoptosis in ovarian follicles.
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    ThesisItemOpen Access
    EFFECT OF PROBIOTICS ON PERFORMANCE, LIPID PROFILE AND IMMUNE STATUS IN BROILER CHICKEN
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-12) VINAYA SREE, C; GUPTA, P.S.P(MAJOR); KONDAL REDDY, K; Gopala Reddy, A; Nagalakshmi, D
    ABSTRACT: The effect of feeding probiotics on growth performance, blood constituents and immune parameters in broiler chicks was studied. An experiment of 42 days duration was conducted 525 day-old broiler chicks, which were randomly allotted to 7 dietary treatments. Each treatment contained 75 chicks. The 7 dietary treatments were control (no probiotic) and other 6 diets were the control diet supplemented with various probiotics viz., Pediococcus acidilactici (MTCC, Chandigarh), Pediococcus acidilactici (NCIM, Pune), Saccharomyces cerevisiae, Saccharomyces boulardii 50 % + Pediococcus acidilactici (NDRI) 50%, Saccharomyces boulardii, Lactobacillus reuteri @ of 109 CFU/gm. Data was collected for body weight, daily weight gain, feed intake and feed conversion ratio during the experimental period (0 – 42 d of age). On day 28 and 42, concentration of proteins, albumin, globulin, albumin/globulin ratio, cholesterol and triglycerides in serum, red blood cells (RBC), haemoglobin, packed cell volume (PCV) and white blood cells (WBC) in chick’s blood were evaluated. Body weight gain of broilers was influenced by inclusion of probiotics during over all period and groups supplemented with Pediococcus acidilactici, (MTCC, Chandigarh), Saccharomyces cerevisiae and combination of Saccharomyces boulardii 50 % and Pediococcus acidilactici 50% recorded the highest body weight amongst the experimental groups at the end of 6th week. Supplementation of probiotics as single or as their combination did influence feed intake in broilers, but had significant effect on feed conversion ratio of broilers for over all period. Livability was not influenced by various probiotics supplementation. The concentration of total proteins and globulins were significantly (P<0.05) higher in all the probiotic supplemented groups and Lactobacillus reuteri recorded the highest protein and globulin concentration. A/G ratio were significantly (P<0.05) lower in all groups while albumin concentration did not reveal any significant changes among the groups. Supplementation of probiotics had profound effect on the serum lipid profile, both serum cholesterol and triglycerides level was significantly (P<0.05) lowered with either of the probiotic supplementation. Mean TEC (106/cmm) was significantly (P < 0.05) lower in control group in comparison to other groups supplemented with probiotics and highest TEC was found in groups Saccharomyces cerevisiae, Lactobacillus reuteri and Saccharomyces boulardii. The mean mean TLC (103/cmm) was highest with Pediococcus acidilactici (MTCC, Chandigarh), Pediococcus acidilactici, (NCIM, Pune) and Saccharomyces cerevisiae. The probiotics Saccharomyces cerevisiae and Saccharomyces boulardii revealed highest Hb (g/dl) at the end of experimental period. The PCV (%) showed a significant (P < 0.05) decrease in control in comparison with that of probiotic fed groups at the end of 6th week and highest PCV (%) was observed with Saccharomyces cerevisiae and Saccharomyces boulardii. The humoral immune response to new castle disease virus at 28 days of age significantly (P<0.05) increased with the inclusion of probiotics. At the age of 42 days, HI titers were significantly (P<0.05) higher in all diets supplemented with probiotic than control. Supplementation of probiotic influenced the faecal pH which significantly (P<0.05) reduced in all dietary treatment groups compared to the control diet. Supplementation of probiotics in all dietary treatment groups lowered the faecal coliform count. The present study indicated that supplementation of probiotics @ (1×109 cfu/g) in diets of broiler diets improved growth, lowered serum lipid profile, increased serum protein profile, haematological constituents, faecal pH and faecal coliform count and better immune responses.
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    ThesisItemOpen Access
    IN VITRO PRODUCTION OF MEIOTICALLY COMPETENT OOCYTES FROM GOAT PREANTRAL FOLLICLES
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2013-09) SHANKARAIAH, PAKALA; SWATHI, B(MAJOR); SRINIVASA PRASAD, Ch; ARUNA KUMARI, G
    ABSTRACT : A series of experiments were carried out with different combinations of various growth factors and hormones like Growth Hormone (GH), Insulin like Growth Factor-I (IGF-I), Epidermal Growth Factor (EGF), Thyroxine (T4) and Follicle Stimulating Hormone (FSH) in culture medium to support growth and development of goat preantral follicles in vitro for a period of eight days and then the oocytes retrieved from cultured preantral follicles were kept for in vitro maturation in four different protocols of IVM media. The effect of Growth Hormone (GH), Insulin like Growth Factor-I (IGF-I), Epidermal Growth Factor (EGF), Thyroxine (T4) and Follicle Stimulating Hormone (FSH) in different combinations was investigated on in vitro development of goat preantral follicles (PFs). Tissue culture medium 199 (TCM 199M) supplemented with Sodium Pyruvate, L-Glutamine, Ascorbic acid, Cysteine and Gentamicin sulfate was used as control medium. Goat PFs (diameter ranging from 150-400μ) were cultured for a period of eight days in different combinations of above mentioned hormones and growth factors. The development of the cultured PFs was assessed by the proportions of follicles exhibiting growth, increase in follicular diameter, antrum formation and extrusion of oocytes in vitro. After eight days of culture period, oocytes were retrieved from different treatments of preantral follicles (PFs) and kept for in vitro maturation in incubator at 38.5oC, 5% CO2 in different IVM media. After 27 hours of in vitro maturation period, oocytes were denuded and evaluated for nuclear status by staining with propidium iodide. In Experiment–1: Based on the previous studies in this laboratory the best concentrations were taken for different growth factors and hormones and evaluated the effect in different combinations of those growth factors and hormones on in vitro development of preantral follicles (PFs). PFs were cultured in various combinations in total of five treatments (T1- T5) (including control) at a concentration of GH (2 mIU/ml), IGF-I (20 ng/ml), EGF (50 ng/ml), Thyroxine (1μg/ml) and FSH (2 ng/ml) supplemented to control media. The proportion of PFs exhibiting growth was highest (98.00±0.87) when cultured in T4+FSH+GH+EGF (T3) supplemented medium but this was not statistically significant to those cultured in other treatments. However, the proportion of PFs exhibiting antrum formation (89.33±1.86) and extrusion of oocyte (15.55±2.09) were also highest when the medium was supplemented with T4+FSH+GH+EGF (T3) with a significant difference with follicles cultured in other treatments. The proportion of PFs showing increase in diameter (42.66±2.06 μ) was highest in T4+FSH+GH+EGF (T3) supplemented medium and this was significantly different to those cultured in treatment groupsT1 (T4+FSH), T2 (T4+FSH+EGF) and T5 (control). However, there was no significant difference with T4+FSH+EGF+IGF (T4) Experiment–2: Based on the results from experiment -1, experiment -II was designed to investigate the maturation status of the in vitro mature oocytes in different IVM media. After culture period of 8 days, oocytes were retrieved from cultured preantral follicles (T1-T5), were kept for in vitro maturation for a period of 27hrs in different IVM media (IVM-I, IVM-II, IVM-III and IVM-IV) and antral oocytes were kept for IVM as control-II in different IVM media. Among in vitro maturation protocols of our study the proportion of oocytes exhibiting M-II stage were maximum in IVM-III. And among treatments T3 (T4+FSH+GH+EGF) had a highest percentage of M II stage in all IVM media and these results were statistically different (P ≤ 0.05) with oocytes of other treatments.
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    ThesisItemOpen Access
    INFLUENCE OF LEPTIN ON IN VITRO DEVELOPMENT OF PREANTRAL FOLLICLES OF SHEEP
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2014-11) Siva Kumar, A.V.N.; Rao, V.H(MAJOR); Punya Kumari, B
    ABSTRACT : The role of human and ovine leptin on in vitro development of preantral follicles (PFs’) isolated from sheep ovaries was studied. Experiment I, was conducted to standardize the optimum concentration of human leptin for the growth of PFs’. The parameters set for the evaluation of the growth of PFs’ were increase in diameter, proportion of PFs’ exhibiting growth, antrum formation and maturation of oocytes to M II stage. All these parameters were higher in the leptin (10ng/ml) supplemented medium. Experiment II was conducted with the optimum level of leptin (10ng/ml) derived in experiment I to evaluate the effect of human or ovine leptin @10ng/ml in combination with various growth factors viz., TCM 199 B with FSH, T4, IGF-I and GH. Two controls viz., TCM 199 B and standard medium i.e., TCM 199 B supplemented with FSH (2ug/ml), T4 (1μg/ml), IGF-I (10ng/ml) and GH (1mIU/ml) were used. In the standard medium supplemented with human leptin, the proportion of PFs’ exhibiting growth (66.14±3.9), average increase in the diameter (11.1±0.87 μ), antrum formation (51.5±5.33) and maturation of oocytes to M II stage (24.1±3.7) were higher compared to the standard medium and standard medium supplemented with ovine leptin (10ng/ml). But there was no significant difference between the standard medium and the standard medium supplemented with ovine/human leptin. It is concluded that (i) the optimum dose of leptin for the growth of sheep PFs’ in vitro was 10ng/ml and (ii) human or ovine leptin supported similar growth of PFs’ cultured in the standard medium.