Arunakumari G.,Sv Veterinary University |
Shanmugasundaram N.,Sv Veterinary University |
Shanmugasundaram N.,University of Illinois at Urbana - Champaign |
Rao V.H.,Sv Veterinary University
Theriogenology | Year: 2010
Sheep preantral follicles (PFs) measuring 250-400 μm in diameter were cultured for six days in serum-free media supplemented differently with growth factors and hormones. Subsequently, oocytes from the cultured follicles were subjected to an additional 24 h of in vitro maturation (IVM) followed by in vitro fertilization (IVF) and embryo culture for 6 days. Five different experiments were conducted. In the first experiment individual concentrations of Insulin-Transferrin-Selenite (ITS), Insulin-like growth factor-I (IGF-I), Transforming growth factor-beta (TGF-β), Insulin (INS), and Growth hormone (GH) that supported the best in vitro development of the PFs were determined. The influence of different combinations of the above hormones and growth factors at their best concentrations as determined in the first experiment was investigated in the second experiment. In the third experiment the best combinations of the growth factors and hormones obtained in the second experiment were additionally supplemented with Thyroxin (T4) and follicle stimulating hormone (FSH) and the influence on in vitro development of the PFs was studied. In the fourth experiment, two methods of culturing PFs-micro drops and agar gel embedding-were compared. In the fifth experiment oocytes from cultured PFs were subjected to IVF and in vitro development of the resulting embryos was followed to the blastocyst stage. Based on the proportion of the PFs exhibiting growth, mean increase in diameter, proportions of PFs developing antrum, ovulations in vitro and oocytes maturing to M-II stage, 1% ITS, 10 ng/mL each of IGF-I, and Insulin and 1 mIU/mL of GH were found to support the best development of sheep PFs. However, the oocytes from PFs cultured in any concentration of TGF-β failed to mature to M-II stage. Similarly, among the combinations studied, IGF-I+GH was found to be the best. In combination with T4 and FSH, IGF-I+GH supported the best development of the PFs. Culture of PFs in micro drops or agar gel supported similarly high development. In vitro fertilization of the oocytes from the cultured sheep PFs resulted in the embryos developing to the morula stage for the first time. © 2010 Elsevier Inc.
Subbaiah K.C.V.,Dravidian University |
Raniprameela D.,Sv Veterinary University |
Visweswari G.,Sv University |
Rajendra W.,Sv University |
Lokanatha V.,Dravidian University
Naturwissenschaften | Year: 2011
The aim of the present study was to investigate the effect of vitamin E on pro/anti-oxidant status in the liver, brain and heart of Newcastle disease virus (NDV) infected chickens. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and the levels of reduced glutathione and malonaldehyde were estimated in selected tissues of uninfected, NDV-infected and NDV∈+∈vit. E-treated chickens. A significant increase in MDA levels in brain and liver (p∈<∈0.05) was observed in NDV-infected chickens when compared to controls. The activities of SOD, CAT, GPx, GR, GST and levels of GSH were significantly (p∈<∈0.05) decreased in brain and liver of NDV-infected chickens over controls. On the other hand, a significant decreased MDA levels and enhanced antioxidant enzyme activity levels were observed in NDV∈+∈vit. E-treated animals compared to NDV-infected chickens. Histopathological studies revealed that liver of NDV infected chicken shows focal coagulation and infiltration of hepatocytes, whereas neuronal necrosis and degeneration of Purkinje cells were observed in brain and moderate infiltration of inflammatory cells was observed in heart. However such histological alterations were not observed in NDV∈+∈vit. E-treated animals. The results of the present study, thus demonstrated that antioxidant defense mechanism is impaired after the induction of NDV, suggesting its critical role in cellular injury in brain and liver. Further, the results also suggest that vitamin E treatment will ameliorate the antioxidant status in the infected animals. The findings could be beneficial to understand the role of oxidative stress in the pathogenesis of NDV and therapeutic interventions of antioxidants. © 2011 Springer-Verlag.
Lakshminarayana B.N.V.,University of Veterinary and Animal Sciences |
Praveen Chakravarthi V.,Sv Veterinary University |
Brahmaiah K.V.,University of Veterinary and Animal Sciences |
Rao V.H.,Sv Veterinary University
Small Ruminant Research | Year: 2014
A consistent observation during in vitro culture of preantral follicles (PFs) of mammals, was that the rates of in vitro maturation and embryogenesis of the oocytes obtained from the cultured PFs was low relative to the oocytes obtained from antral follicles. It was hypothesized that the suboptimal development potential of the oocytes from cultured PFs may be related to the altered expression patterns of developmentally important genes. Expression of P450 aromatase gene is needed for the synthesis of steroid hormones in the ovarian follicles as they develop from preantral to Graafian follicle stage. The present study was undertaken to compare the quantitative expression of P450 aromatase gene in the sheep oocytes and granulose cells obtained from in vivo grown and cultured ovarian follicles from preantral to Graafian follicle stages.P450 aromatase expression was found in granulose cells obtained from all the stages of in vivo grown follicles. In the in vitro cultured follicles aromatase expression was found in the granulose cells from 2 day cultured follicles only. Similarly the P450 aromatase expression was observed in the oocytes from all the in vivo grown stages of follicles but from none of the in vitro grown stages.It is concluded that the culture system for sheep PFs decreased P450 aromatase expression leading to compromised development potential of the oocytes. Present results provide support to the hypothesis that the compromised development potential of the oocytes from cultured ovarian follicles is related to aberrant expression of developmentally important genes. © 2013 Elsevier B.V.
Chakravarthi V.P.,Sri Venkateswara University |
Kona S.S.R.,Sv Veterinary University |
Siva Kumar A.V.N.,Sv Veterinary University |
Bhaskar M.,Sri Venkateswara University |
Rao V.H.,Sv Veterinary University
Small Ruminant Research | Year: 2016
Expression of gap junction genes CX32 and Cx43 was studied in different stages of in vivo and cultured ovarian follicles in sheep. In the in vivo grown follicles, CX43 expression in the cumulus cells did not change with development but in the oocytes a significant decrease was noted in the early antral follicles. Although CX32 expression in the cumulus cells appeared to decrease continuously, it was significant only from early antral to antral follicles. However, Cx32 expression in oocytes showed an increasing pattern although the increase from early antral to antral stage was not significant. In the cultured follicles CX43 expression in cumulus cells decreased significantly from preantral to early antral follicles, then increased significantly at the antral stage and decreased again in the large antral follicles. The pattern of CX32 expression was similar except for the significant decrease observed with CX43 expression in the large antral follicles. In the oocytes CX43 expression increased significantly from preantral to early antral stage, and decreased significantly at antral and large antral stages. On the other hand CX32 expression increased significantly from early antral to antral follicle stage and then decreased significantly in the large antral follicles. Subsequent to in vitro maturation for 24 h of COCs from in vivo grown large antral follicles, CX43 expression was supressed both in the cumulus cells and oocytes but CX32 expression was compromised only in the oocytes. In the similarly treated COCs’ from in vitro grown large antral follicles, CX43 expression was stimulated both in the cumulus cells and oocytes but CX32 expression was augmented only in the cumulus cells. It is concluded that (i) the gap junction genes follow a stage specific pattern of expression during ovarian follicular development and (ii) in vitro culture adversely influenced the expression of the gap junction genes. © 2016 Elsevier B.V.