Proceedings of the 5th International Symposium on EQUINE ...

Equine Embryo TransferTHE DEVELOPMENT OF BLASTOCYSTS AFTERINTRACYTOPLASMIC SPERM INJECTION OF EQUINEOOCYTESX. Li, L. H. A. Morris and W. R. AllenEquine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, Suffolk CB8 9BH, UKINTRODUCTIONHorse oocytes obtained from abattoir ovaries andcultured in vitro can complete nuclear maturation byreleasing <strong>the</strong> first polar body <strong>of</strong> Metaphase II (MII)after 15–40 h <strong>of</strong> culture (Zhang et al. 1989; Hinrichset al. 1993). Although <strong>the</strong>se MII oocytes havecompleted nuclear maturation, it remains difficult t<strong>of</strong>ertilise <strong>the</strong>m by ei<strong>the</strong>r IVF or ICSI and to induce<strong>the</strong>ir fur<strong>the</strong>r development to <strong>the</strong> blastocyst stageduring subsequent culture in vitro (Choi et al.1994;Dell’Aquila et al. 1997; Li et al. 2000).The importance <strong>of</strong> <strong>the</strong> oviduct in gametematuration and fertilisation has been established(Hunter 1994), and co-culture with oviductepi<strong>the</strong>lial cells is used increasingly to supportoocyte maturation and early embryonicdevelopment in vitro. In <strong>the</strong> present study weinvestigated <strong>the</strong> influence <strong>of</strong> oviduct epi<strong>the</strong>lialcells (OEC) on <strong>the</strong> maturation, fertilisation andsubsequent development potential <strong>of</strong> abattoirderivedequine oocytes cultured in vitro after ICSI.MATERIALS AND METHODSHorse ovaries were obtained from twoslaughterhouses and transported to <strong>the</strong> laboratorywithin 24 h. Cumulus oocyte complexes (COCs)were matured in vitro for 28–30 h, with orwithout OEC, in TCM199 medium containing20% FBS (v:v), 10 µg/ml FSH, 5 µg/ml LH and 1mg/ml oestradiol, at 38ºC in 5% CO 2 -in-air.Three types <strong>of</strong> spermatozoa were prepared forICSI; washed, acrosome-reacted and predecondensedspermatozoa.The injection pipette containing <strong>the</strong>spermatozoon was pushed through <strong>the</strong> zonapellucida and plasma membrane so that <strong>the</strong>spermatozoon could be injected directly into <strong>the</strong>cytoplasm. The injected oocytes were <strong>the</strong>nactivated by immersing <strong>the</strong>m in STALP solutioncontaining 5 µM ionomycin and 1% ethanol for 5min. After both ICSI and <strong>the</strong> activation procedure,groups <strong>of</strong> 5–10 oocytes were cultured for 7–9 daysin DMEM medium with cumulus cells monolayer(CCM) at 38ºC in an atmosphere <strong>of</strong> 5% CO 2 -in-air.Two-cell stage embryos were stained withHoechst 33342 (Sigma) in PBS solution (5 µg/ml)for 10 min at 38ºC before being examined underultraviolet light (excitation wavelength 330–380nm) using a fluorescent microscope.CONCLUSIONSThe mammalian oviduct normally provides <strong>the</strong>optimum environment for <strong>the</strong> final maturation <strong>of</strong>TABLE 1: Nuclear maturation <strong>of</strong> horse oocytes cultured in vitro, with and without oviduct epi<strong>the</strong>lialcells (OEC)Maturation Total number Number and (%) <strong>of</strong>medium <strong>of</strong> oocytes MII≠ MIIIVM-1 145 74(51) 71(49)IVM-1/OEC 140 77(55) 53(45)30