Cysteamine, glutathione and ionomycin treatments improve in vitro fertilization of prepubertal goat oocytes.

The aim of this study was to improve in vitro embryo development of prepubertal goat oocytes by studying the effect of adding cysteamine to in vitro maturation medium, glutathione (GSH) to in vitro fertilization medium and ionomycin to the sperm capacitation medium. In experiment 1, we analysed the effect of 1 mM GSH added to fertilization medium of oocytes matured with 400 microM cysteamine. The control group were oocytes without cysteamine and GSH. In experiment 2, oocytes matured and fertilized in the presence of 400 microM cysteamine and 1 mM GSH, respectively, were inseminated with spermatozoa treated with ionomycin or heparin. In experiment 1, the percentages of total and normal fertilized oocytes were significantly higher for oocytes supplemented with cysteamine and GSH (40.26% and 30.20%, respectively) than for oocytes from the control group (16.66%, and 10.61%, respectively). The percentage of total embryos obtained after 7 days of culture was significantly higher in the group supplemented with cysteamine and GSH (30.62%) than in the control group (8.09%). In experiment 2, percentages of total and normal fertilized oocytes were significantly higher for the group of spermatozoa capacitated with ionomycin (52.21% and 37.17%, respectively) than with heparin (38.62% and 28.35%, respectively). After 7 days of culture, total embryo rate was significantly higher in the group of sperm capacitated with ionomycin (44.91%) than with heparin (38.69%). However, the percentage of embryos developed to the blastocyst stage was not affected by any of the treatments studied.

Effect of the addition of glutathione and glucose to the culture medium on embryo development of IVM-IVF prepubertal goat oocytes.

Our previous studies have shown that larger and more competent oocytes can be selected using the brilliant cresyl blue (BCB) test. The objective of this study was to assess, in BCB-selected oocytes, the effect on the embryo development of prepubertal goat oocytes of the addition to in vitro culture (IVC) medium of either glutathione (GSH) alone or GSH in combination with glucose. Oocytes were exposed to 26 mM BCB and were classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocyteswithout blue cytoplasm or growing oocytes (BCB-). Oocytes were matured in TCM-199 with 100 microM cysteamine. Presumptive zygotes were cultured in synthetic oviductal fluid (SOF) in the presence or absence of 1 mM glutathione (experiment 1) for 7 days (8 days post-insemination, p.i.). In experiment 2 we tested the addition to culture of 2.78 mM glucose at day 5 p.i. BCB+ oocytes showed higher percentages of nuclear maturation than the BCB- and control groups (82.6%, 55.7% and 74.7%, respectively). The percentage of polyspermic oocytes was higher in BCB- than BCB+ oocytes. Supplementation of SOF medium with 1 mM GSH did not affect embryo development but the percentage of total embryos developed after culture was higher in BCB+ oocytes than in BCB- oocytes independently of the GSH supplementation. Glucose, alone or with GSH, added at 5 days p.i. did not affect embryo development. In conclusion, prepubertal goat oocytes were unable to develop beyond the 8-cell stage embryo under the culture conditions in this study.