Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos.

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 degrees C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p < 0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p < 0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p > 0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p < 0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p > 0.05) among methods 1, 3 and 4, but method 2 produced a higher (p < 0.05) blastocyst rate than method 3.