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2010 - 20154
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Subject: Animal Physiology ×
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    ThesisItemOpen Access
    VALIDATION OF REFERENCE GENES FOR THE QUANTITATIVE GENE EXPRESSION STUDIES IN THE OVARIAN FOLLICLES OF SHEEP
    (Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2012-12) VASANTHA SESHU KUMARI, I (Major); RAO, V.H (Major); SIVA KUMAR, A.V.N; ADILAXMAMMA, K
    ABSTRACT : To confirm if the poor development of oocytes from cultured preantral follicles (PFs’) of sheep was indeed due to the aberrant expression of developmentally important genes, it is necessary to study the expression patterns of these genes in the cultured PFs’. While absolute and relative qRT-PCR are the two related techniques, relative quantification employing reference genes is the widely used method. Reference genes are assumed to be stably expressed in all tissues and under varied experimental conditions. However, such an assumption was frequently unfitting and therefore it was repeatedly emphasized that the reference genes need be validated for different tissues and experimental conditions. Accordingly in the present study six of the commonly used reference genes viz., ACTB, GAPDH, HPRT1, 18S rRNA, PPIA and RPLPO were validated for the gene expression studies in in vivo and in vitro grown PFs’ of sheep. The expression pattern of these reference genes in the granulosa cells and oocytes was studied by qRT-PCR in different development stages of in vivo and in vitro grown ovarian follicles. Data was analysed by RefFinder which employs four different software viz., ΔCt, Best keeper, Norm Finder and geNORM simultaneously to rank the genes according to their stability of expression and also suggests comprehensive ranking based on the results obtained in the four different software together. From the results it is concluded that for the gene expression studies in sheep (i) granulosa cells from in vivo grown follicles RPLPO, GAPDH and PPIA, (ii) oocytes from in vivo grown follicles PPIA, HPRT1, RPLPO, (iii) granulosa cells from in vitro grown follicles PPIA, 18S rRNA, RPLPO, (iv) oocytes from in vitro grown follicles GAPDH, HPRT1, PPIA and (v) ovarian follicles as a whole whether in vivo or in vitro grown GAPDH, PPIA and HPRT1 may be used as reference genes.
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    ThesisItemOpen Access
    PHYSIOLOGICAL CHARACTERIZATION OF PUNGANUR CATTLE
    (Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2011-09) RAMBABU NAIK, B; RAO, V.H(Major); SIVA KUMAR, A.V.N.; RAVI, A; BRAHMAIAH, K.V.
    ABSTRACT : The present study was conducted at Livestock Research Station, Palamaner, Chittoor district to establish base line physiological, hematological and biochemical values in Punganur cattle of different age groups (bulls, cows, young bulls, heifers, male and female calves) during different seasons (monsoon, winter and summer) and also for hormones like estrogen and progesterone levels during estrous cycle in Punganur cows. The mean rectal temperature (oF), respiration, pulse and heart rate (per min.) were 99.93 to 100.92, 21.16 to 27.98, 59.79 to 63.56 and 62.94 to 67.95, respectively, that were significantly higher (P<0.01) in young animals than in adult cows and bulls and also during summer than during monsoon and winter seasons. The mean RBC count (7.50 to 8.68 millions/cmm) and the mean WBC count (8.69 to 11.27 thousands/cmm) differed significantly (P<0.01) among the age groups and were higher during summer than either during monsoon or winter season. The Hb content (10.51 to 11.39 g/dl) was not significantly different among the age groups but was significantly higher (P<0.01) during summer (12.11 g/dl) than during either monsoon (11.02 g/dl) or winter (10.33 g/dl) seasons. The mean PCV (%) in Punganur cattle of different age groups (35.09 to 39.87%) and also during monsoon (36.53%), winter (35.50%) and summer (37.45%) seasons were not significantly different. The mean ESR (mm/hr) in Punganur cattle of different age groups (3.13 to 3.46) was not significantly different, but was significantly (P<0.01) higher during summer (3.42 mm/hr) than during winter (3.11 mm/hr). The DLC count (%) among different age groups of Punganur cattle was significantly different (P<0.01) for neutrophils, eosinophils, basophils, lymphocyte except for monocytes. Neutrophils and lymphocytes were significantly higher (P<0.01) during summer, eosinophils during monsoon, and basophils during monsoon and winter while monocytes count was significantly higher (P<0.01) during winter than during other seasons. `The mean values (%) for the different leucocyte count were in the range of 25.57 to 28.24 (neutrophils), 4.28 to 6.03 (eosinophils), 0.18 to 0.58 (basophils), 6.45 to 6.98 (monocytes), 59.49 to 62.00 (lymphocytes) for Punganur cattle of different age groups. The mean calcium level (mg/dl) of bulls was significantly higher (P<0.01) than other age groups with the values ranging from 6.95 to 9.83 and was also significantly higher (P<0.01) either during monsoon or summer than during winter. The mean phosphorus level (mg/dl) in Punganur cattle was not significantly different among the age groups or during different seasons with the value ranging from 2.96 to 3.43 for different age groups. The estrogen level (ng/ml) in Punganur cows during estrous cycle was significantly higher (P<0.01) on ‘0’ day (20.24) and decreased upto 15th days (12.36) and thereafter gradually increased to a level of 22.58 on day ‘00’. The mean progesterone level (ng/ml) in Punganur cows during estrous cycle increased significantly (P<0.01) upto 15th day of estrus cycle and gradually decreased thereafter with the values ranging from 0.43 (‘0’ day) to 10.66 (15th day) and from 8.64 (18th day) to 0.34 (day 00).
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    ThesisItemOpen Access
    STUDIES ON EXPRESSION OF INSULIN LIKE GROWTH FACTOR BINDING PROTEIN 3 (IGFBP 3) GENE IN CULTURED AND IN VIVO DEVELOPED PREANTRAL FOLLICLES IN SHEEP
    (Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2010-10) SIVA SAGAR REDDY, K; RAO, V.H (Major); SIVA KUMAR, A.V.N; PADMAJA, K
    ABSTRACT: Expression of Growth Differentiation Factor 9 (GDF 9) and Insulin like Growth Factor Binding Protein 3 (IGFBP 3) genes was studied in oocytes and granulosa cells isolated from various in vivo and in vitro follicular development stages in sheep. In the present study, the expression of both the genes was highest in the primordial follicles. The expression of GDF 9 in in vivo and in vitro oocytes decreased as the development progressed except in large antral follicles (OA2). Among the granulosa cells, the expression of GDF 9 in in vivo stages decreased continuously. In the in vitro granulosa cell stages, the expression of GDF 9 increased except PFs’ cultured for 6 days (G6). The expression of IGFBP 3 in in vivo and in vitro oocytes increased as the development progressed except in antral follicles (OA1). Among the granulosa cells, the expression of IGFBP 3 in in vivo and in vitro stages increased as the development progressed except PFs’ cultured for 6 days (G6). When the oocytes from large antral follicles (OA2) and 6 day cultured PFs’ (O6) were matured in vitro for 24h, the expression of both the genes decreased. When the granulosa cells from large antral follicles (GA2) and 6 day cultured PFs’ (G6) were matured in vitro for 24h, the expression of both the genes increased. The expression of both the genes increased significantly in the oocytes and granulosa cells from in vitro cultured PFs’ than their corresponding in vivo stages
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    ThesisItemOpen Access
    INFLUENCE OF LEPTIN ON THE EXPRESSION OF BCl2 and BAX GENES IN CULTURED OVARIAN FOLLICLES IN SHEEP
    (SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502,A.P, 2015-11) DEEPA, P; SIVA KUMAR, A.V.N (Major); Rao, V.H; Padmaja, K
    ABSTRACT: To ascertain whether the support provided by Leptin to the PFs’ in culture (Kamalamma et al., 2015) was due to its ability to modulate apoptosis related genes, the present study is undertaken. Accordingly in the present study expression levels of two test genes BCl2 (antiapoptotic), BAX (apoptotic) and three reference genes viz., 18S rRNA, HPRT1, RPLPO were studied in the Cumulus cells and Oocytes during in vivo and in vitro folliculogenesis in sheep. Sheep ovaries were collected from the local slaughter house. The Cumulus cells and Oocytes from different development stages of ovarian follicles i.e., preantral, early antral, antral and large antral follicles were isolated. Preantral follicles (PFs’) isolated from the ovaries were cultured in a medium containing 10 ng/ml of recombinant human Leptin for 3 minutes, 2, 4, or 6 days to obtain cultured preantral, early antral, antral and large antral follicles respectively. Furthur some COCs in in vivo and in vitro grown large antral follicles were matured in vitro for 24h. The expression pattern of the test and reference genes (18SrRNA, HPRT1, RPLPO) in the Cumulus cells and Oocytes was studied separately at the different development stages mentioned earlier by Quantitative Reverse transcription Real time PCR (qRT-PCR). Quantitative expression of BCl2 gene in the Cumulus cells isolated from different in vivo grown ovarian follicles was significantly lower in the pre antral and early antral follicles than corresponding stages of follicles cultured in Leptin. However, the expression was significantly higher in the in vivo grown antral, large antral and COCs from large antral follicles matured in vitro for 24h. Quantitative expression of BCl2 in the Oocytes isolated from the pre antral follicles and COCs in large antral follicles matured in vitro for 24h was significantly lower than their corresponding Leptin cultured stages. However, there was no significant difference in the BCl2 expression in the Oocytes in early antral, antral and large antral follicles compared to their corresponding Leptin cultured stages. Quantitative expression of BAX in the Cumulus cells isolated from in vivo grown ovarian follicles was significantly lower in the preantral follicles and Cumulus cells from COCs after in vitro maturation for 24h than their corresponding Leptin cultured stages. However, the Cumulus cells from in vivo antral follicles exhibited a significantly higher level of expression than corresponding Leptin cultured follicles. Quantitative expression of BAX in the Oocytes isolated from the in vivo grown antral follicle and Oocytes from COCs after in vitro maturation for 24h was significantly lower than the corresponding in vitro stage. However, Oocytes from large antral follicles expressed BAX to a significantly higher level than corresponding in vitro stage. The expression pattern of BAX in the Oocytes isolated from the different in vivo grown ovarian follicles was similar to the corresponding in vitro grown stages in early antral and large antral follicles. From the present study it is concluded that supplementation of Leptin (10 ng/ml) in the culture medium induced a different quantitative expression pattern of BCl2 in Cumulus cells and Oocytes than in vivo grown ovarian follicles. Leptin inclusion in the culture medium was able to simulate the in vivo pattern of BAX gene expression in the Cumulus cells and Oocytes at some but not all of the development stages.