The objective of this study was to investigate the influence of sperm mitochondrial destruction on sperm head decondensation, male pronuclear formation, cleavage and embryonic development. In the study two models were used:heterologous (hamster ICSI assay: human sperm injected into a hamster oocyte) for evaluation of sperm head decondensation and pronuclear formation, and homologous (mouse model) for the study of fertilisation and development. Destruction of mitochondria of the sperm was achieved by exposure to cyanide, a respiratory poison. Rhodamine 123 was used to evaluate the functional integrity of mitochondria. Sperm head decondensation was found to be not statistically significantly affected by mitochondrial damage (p = 0.8), with 62.8% and 67.9% condensa tion in the experimental and control groups respectively. Male pronucleus formation was seen in 40.2% and 44.4% of the injected oocytes in the experimental and control groups respectively. In the mouse experiments 45.5% and 49.7% of the injected oocytes were fertilised in the mitochondria-damaged and live-intact sperm groups respectively(p = 0.53)Development to blastocyst was achieved in 53.5% and 59.4% of the experimental and control groups respectively; the difference was not significant (p = 0.71). Inner cell mass (ICM) cell number were 15.7 ± 4.02 and 43.1 ± 11.3 respectively in the mitochondria damaged group; the equivalent numbers were 14.12±4.12 and 39.3 ± 12.6 in the control group. However, the differences in ICM and total cell counts between these two groups were not significant. Of the blastocysts transferred to pseudopregnant mice, 51.3% (20/36) implanted and 33.4% (12/36) developed to live fetuses in the mitochondria-damaged group. These rates were 60.5% (23/38) and 39.5% (15/38) in the control group. In conclusion, this study shows that functional integrity of thesperm mitochondria is not necessary in the process of fertilisation and development when the sperm is deposited into the ooplasm. Fertilisation and development can be achieved by injection of sperm at the very early stage of necrosis in which only the mitochondria have been destroyed and the rest of the cell including the plasma membrane is still intact.

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.