Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24-26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18-20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.

Gender preselection in cattle with intracytoplasmically injected, flow cytometrically sorted sperm heads.

We investigated the development to the blastocyst and subsequent live-offspring stages of in vitro-matured bovine oocytes intracytoplasmically injected with flow cytometrically sorted bull sperm heads. Bull sperm heads, prepared by ultrasound sonication, were distinguished and sorted on the basis of their relative DNA contents using a flow cytometer/cell sorter modified for sorting sperm. By fluorescence in situ hybridization, the proportion of sperm confirmed as having Y specific DNA in the fraction sorted for the Y sperm was 82%. Injection with single sorted sperm heads of in vitro-matured oocytes (cultured for 24 h) resulted in 46.6% cleavage and 6.9% blastocyst development rates. Embryo transfer of 48 blastocysts (Days 7-8) to recipients (one per recipient) resulted in 20.8% pregnancy and 20.8% normal live offspring production rates. The birth of 8 male and 2 female calves represents an 80% sex preselection accuracy rate.