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In vivo and in vitro studies of early foliculogenesis in bovines


This study aims to evaluate different in vitro and in vivo culture techniques regarding growth of preantral follicles from bovines. Thus, the experimental design will carry out by in vitro culture (Phase 1) based on well successful protocol of preantral follicles culture and testing different resources of macromolecules; In vivo culture of preantral follicles (Phase 2) will carry out by xenografting with or without hormonal stimulation of recipients and Phase 3 by comparison between the best results obtained from in vivo (phase 1) and in vivo (phase 2). For experiments, a total of 40 fetal ovaries, from a local slaughterhouse, will be collected, processed at laboratory and cutted them into small cortical pieces, measuring approximately 1mm3. The preantral follicles will be classified according their diameter and morphology and equally distributed for treatments. For in vitro cultures, the cortical pieces will be cultured at controlled atmosphere (5%CO2 in air, 39°C), the culture medium will be dMEM (300 mOsm/L, pH 7,2) supplemented with antibiotics, antimicotic, ITS, sodium pyruvate, glutamin, hipoxantin, dAMPc, bFSH, and IGF-I. Depending of treatment, will be added BSA (1%), FCS (10%) or PVA (1%). For in vivo culture, ovarian cortical pieces will be introduced under renal capsule of immunosuppressed female mice by xenografting technique, subdivided into three groups (control, FSH or eCG stimulated) and subsequently evaluated. The follicles, obtained after cultures will be evaluated by individual growth on days zero and end of culture and their integrity by inverted microscope throughout culturing period, histology and electron microscopy in all cultures. Presumptive antral follicles that measuring between 2 and 6 mm will be puctured and their oocytes submitted at in vitro production procedures. The percentage of normally and atretic follicles will be compared by chi-square test (p<0,05). In vitro and in vivo growth will be evaluated by regression analysis inside treatments and, between treatments, by ANOVA's test followed by Tukey's test (p<0,05).Key-words: Ovary, preantral follicles, xenografting, bovine fetus, mouse. (AU)

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