• Journal of Embryo Transfer
• /
• v.13 no.3
• /
• pp.227-233
• /
• 1998

The aim of these experiments was to investigate the effects of oviduct epithelial cells on bovine in vitro fertilization. Oviduct epithelial cell monolayers (OEC) on the 4-well dish were prepared according to general procedures. Monolayers were formed within 5days. The medium for OEC culture (TCM199 with 10% FBS) was replaced with IVF-TALP 2h before each experiment. Macromolecules/proteins from oviductal conditioned medium (OM) were recovered by ultrafiltration, which desalted and concentrated macromolecules greater than 5kDa, and this OM were added to W medium (experiment 1). The cleavage rate in OM+OEC group was significantly higher than in OM group (p〈0.01). In this experiment 2, oocytes were inseminated on OEC with sperm which had been pre-incubated with OEC for 0 or 4h before insemination. In this experiment, oocytes were exposed to sperm only 8 h for clarifying the effect. After insemination, oocytes were cultured in CRlaa. At 42 h post insemination, oocytes were denuded and examined for evidence of cleavage. The cleavage rates of oocytes which were inseminated with OEC treated sperm for 4 h were significantly higher than those of the other group (p〈0.01). In conclusion, sperm released from OEC have more fertilizing ability than those before attachment.

• Journal of Embryo Transfer
• /
• v.9 no.2
• /
• pp.145-152
• /
• 1994

The suitable electric stimulation is essential for activation and fusion of oocytes before or after nuclear transplantation The present study was undertaken to determine the optirnal condition for the parthenogenetic activation of in vitro rnatured(IVM) bovine oocytes by electric stimulation. Different direct current(DC) electric voltage of 1.0, 1.5 and 2.0 kV/cm and pulse duration of 30, 60 and 120 $\mu$sec were applied to the JVM nocytes in 0.3 M mannitol solution containing each 100 $\mu$M CaCl$_2$ and MgCl$_2$. IVM occytes at 24, 28 and 32 hours Post-maturation(hpm) were also electrically stimulated at 1.5 kV /cm, for 60 $\mu$ sec. The stimulated nocytes were then co-cultured in TCM-199 solution containing 10% fetal calf serum with bovine oviductal epithelial cells for 7~9 days in a 5% $CO_2$ incubator at 39$^{\circ}C$ ~ Their activation and in vitro development to morula and blastocyst were assessed under an inverted microscope. The higher activation rates 62.8 and 63.4% and in vitro de- velopment rates to morula and blastocyst 5.1 and 10.9% were shown in the oocytes stimulated at the voltage of 1.0 and 1.5 kV/cm than 2.0 kV/cm, respectively. No signifi- cantly(P<0.05) different activation rate was shown in JVM oocytes stimulated for 30, 60 and 120 $\mu$sec, but developmental rates to morula and blastocyst was significantly(P<0.05) higher in the oocytes stimulated for 30 $\mu$sec(6~3%) and 60 $\mu$sec(10~0%) than 120 $\mu$sec(0~ 0%). The aged oocytes at 28 and 30 hpm showed significantly(P<0.05) higher activation rates(72~7 and 79.7%) than the oocytes at 24 hpm(50~9%)~ Also, their developmental rates to morula and blastocyst were significantly(P<0.05) higher in the nocytes at 28(14.3%) and 32 hpm(15.9%) than 24 hpm(3.6%). From these results, it can be suggested that the optimal electric stimulation for IVM bovine occytes is a DC voltage between 1.0 and 1.5 kV/cm, pulse duration of 30 or 60 $\mu$sec, and the optimal age of IVM oocytes for electric activation is at 32 hpm.