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ThesisItem Open 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, GABSTRACT : 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.