Development of morulae from the oocytes of cultured sheep preantral follicles

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.