INTRACYTOPLASMIC SPERM INJECTION OF IN-VITRO MATURED EQUINE OOCYTES

Intracytoplasmic sperm injection (ICSI) was performed on equine oocyte s matured in vitro. The oocytes were aspirated from abattoir ovaries a nd matured in vitro for 36 h at 38 degrees C. ICSI was performed using frozen/thawed stallion semen after swimup in medium containing human serum albumin. Sperm-injected oocytes were either 1) cultured in vitro for 10, 20, or 72 h; 2) transferred to oviducts of pseudopregnant mic e; or 3) transferred to a synchronized mare after initial in vitro cul ture. The transferred ova were recovered after 72 h, and an ova were s ubsequently fixed, stained, and processed for light and transmission e lectron microscopy. Single pronucleus formation was observed in 2 out of 12 presumptive zygotes 10 h postinjection, at which time abundant c ortical granules were observed in the subplasmalemmal region. Twenty h ours postinjection, however, 2 pronuclei were observed in 6 of 12 inje cted oocytes (fertilization rate 50%), and almost all cortical granule s were released. The cleavage rate in vitro was 16% after 72 h in cult ure, and the most advanced embryo stages obtained were 6- to 8-cell em bryos. The cleavage rate in vivo was very low since only 1 of 10 recov ered had cleaved to the 2-cell stage. Thus, in conclusion, ICSI fertil ization of equine oocytes did result in fertilization, pronucleus form ation, and cortical granule release. However, the observed fertilizati on rate and oocyte activation was not paralleled by substantial cleava ge of the zygotes.