Embryology Research Today is a free monthly online journal that collates and summarizes the latest research about Embryology, including details on stem cells, reproduction, transplants, cloning. | ||||||||
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Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine.Adona PR, Pires PR, Quetglas MD, Schwarz KR, Leal CL ZAB, FZEA, USP, Pirassununga, SP, Brazil. Cyclin dependent kinase inhibitors (CDKIs) may be used for pre-maturation culture, but can accelerate nuclear maturation. The aim of the present research was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at lesser than typically used concentrations on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100muM BLI (B) or 6.25muM BLI+12.5muM ROS (BR) in TCM-199 for 24h. Oocytes were subsequently submitted to in vitro maturation (IVM) in TCM-199+0.5mug/ml FSH, 50mug/ml LH and 10% FCS for another 24h, during which oocytes were fixed every 3h. In Experiment 2, oocytes were submitted to 24h pre-maturation treatments, with the inhibitors being diluted in TCM-199 or DMEM. IVM lasted 21h in the culture media DMEM+0.5mug/ml FSH, 50mug/ml LH, 5% FCS and 50ng/ml EGF. After IVM, oocytes from all groups were fertilized in vitro. Oocytes and sperm (2x10(6) sperm cells/ml) were co-cultured for 18h. Embryos were co-cultured with granulosa cells in CR2aa for 8 days. All cultures were in droplets under oil, at 38.5 degrees C and 5% CO(2) in air. In both experiments, control oocytes (C) were submitted only to IVM. In Experiment 1, at 0h, C and B oocytes were all (100%) at the germinal vesicle stage (GV) of development. BR had fewer GV oocytes (89%, P<0.05). After 3h IVM, B and BR had fewer oocytes in GV (84.7 and 79.6%, P>0.05) than C (100%, P<0.05). At 12h, most oocytes were at intermediate stages (metaphase to telophase I) in all groups ( approximately 80%, P>0.05). After 21 (77-89%) and 24h (85-95%), all groups had similar metaphase II (MII) rates of development (P>0.05). In Experiment 2, cleavage (79-84%, P>0.05) and Day 7 blastocyst rates (26-36%, P>0.05) were similar. After 8 days, the group pre-matured with BR in DMEM had lesser blastocyst rates of development (32.3%) lower than C (40.1%, P<0.05). The other groups were similar to C (35-38%, P>0.05). Hatching rates were similar (10-15%, P>0.05) as were total cell numbers (141-170). In conclusion, BR is less effective in maintaining meiosis block; B and BR accelerate meiosis resumption; and use of pre-maturation medium may affect developmental rates. Published 4 February 2008 in Anim Reprod Sci, 104(3): 389-97.
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