SPERM AND OOCYTE TREATMENTS TO IMPROVE THE FORMATION OF MALE AND FEMALE PRONUCLEI AND SUBSEQUENT DEVELOPMENT FOLLOWING INTRACYTOPLASMIC SPERM INJECTION IN TO BOVINE OOCYTES

This study assessed pronuclear formation, the chromosomal constitution , and the developmental capacity of bovine zygotes formed by intracyto plasmic injection of oocytes with sperm, treated or not with dithiothr eitol (DTT). Oocytes were matured in vitro for 22-24 h and then centri fuged so that sperm, prepared by swim-up in the presence or absence of 5 mM DTT, could be injected into the cleared area of the ooplasm. Inj ected oocytes were activated by treatment with 5 mu M ionomycin (5 min ) and, after a 3-h interval, with 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h. They were then cocultured with bovine oviductal epithelial ce lls in M199, Sperm treatment resulted in a significantly higher propor tion of male pronucleus formation 16 h after injection (40% vs. 11%; p < 0.0001) and a significantly higher rate of blastocyst development ( 24% vs. 10%; p < 0.005). Sixty-one percent of blastocysts produced wit h treated sperm were diploid. Of 12 blastocysts produced with treated sperm and sexed by a polymerase chain reaction, 4 were male and 7 fema le, and in one a definite diagnosis could not be made. Embryo transfer (2 embryos per heifer) resulted in pregnancies in 6 of 16 recipients at Day 49, but none was carried to term. These results show that the e fficiency of bovine intracytoplasmic sperm injection can be improved b y sperm pretreatment with DTT and by oocyte activation with ionomycin plus DMAP, although the developmental capacity of the resulting embryo s remains limited.