From equine oocyte to equine embryo
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke
Equine oocytes, which are collected in vivo by ovum pick-up (OPU) of immature follicles or in vitro from ovaries post mortem, will first be matured. This in vitro maturation (IVM) consists of incubation of the oocytes in culture medium containing follicle stimulating hormone (FSH) in 5% CO2 in air at 38.2°C, and during 24-30h depending on the cumulus morphology (expanded versus compact). Mature oocytes are characterized by expansion of the cumulus cells and extrusion of the polar body. The presence of a polar body will be judged after denuding the oocyte and these mature oocytes can be fertilized by means of intracytoplasmic sperm injection (ICSI). Sperm for ICSI can be fresh or frozen-thawed and the best sperm cells are selected by means of swim-up or gradient centrifugation. For conventional ICSI, a beveled injection pipette is used, the sperm cell is immobilized by crushing of the tail against the bottom of the plate, and the oolemma is penetrated mechanically by aspiration. During piezo-assisted ICSI, the sperm cell is immobilized by piezo-electric pulses and the oocyte barriers are drilled with a blunt pipette. After injection of the sperm cell into the cytoplasm of the oocyte, the presumed zygotes are cultured in DMEM/F12 with 10% fetal calf serum (FCS) in 90% N2, 5% CO2 and 5% O2 at 38.2°C. The blastocyst stage is reached after 7-9 days and these blastocysts can be transferred to a recipient mare.