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

Havemeyer Foundation Monograph Series No. 3FERTILISATION RATES OF IN VITRO MATUREDOOCYTES TRANSFERRED INTO THE OVIDUCT OFINSEMINATED MARESC. C. Love, S. P. Brinsko*, D. D. Varner* and K. HinrichsDepartment <strong>of</strong> Physiology and Pharmacology; *Department <strong>of</strong> Large Animal Medicine and Surgery,College <strong>of</strong> Veterinary Medicine, Texas A & M University, College Station, Texas 77843-4475, USAIn vitro-matured (IVM) oocytes are commonlyused for experimentation involving in vitr<strong>of</strong>ertilisation (IVF). However, <strong>the</strong> fertilisationpotential <strong>of</strong> IVM oocytes is unknown. The majorbarrier to IVF in <strong>the</strong> horse appears to bepenetration <strong>of</strong> sperm through <strong>the</strong> zona pellucida. If<strong>the</strong> zona is opened by microdissection or zonadrilling, IVF rates are high (Choi et al. 1994; Li etal. 1995). It is possible that in vitro maturation <strong>of</strong>oocytes is associated with hardening <strong>of</strong> <strong>the</strong> zonapellucida which precludes penetration <strong>of</strong> sperm.Hardening <strong>of</strong> <strong>the</strong> zona due to premature release <strong>of</strong>cortical granules has been documented duringIVM in o<strong>the</strong>r species under specific cultureconditions. The ability <strong>of</strong> IVM oocytes to allowsperm penetration must be ascertained before <strong>the</strong>ymay be used effectively for research in IVF. Onlyone previous study (Zhang et al. 1989) hasdocumented fertilisation and development <strong>of</strong> IVMoocytes after transfer to mares. In that report, 4transfers were performed. Of 29 oocytestransferred, 5 confirmed blastocysts wererecovered. The objective <strong>of</strong> our study was todetermine <strong>the</strong> in vivo fertilisation rate <strong>of</strong> oocytesmatured in vitro in 3 different media andtransferred to <strong>the</strong> oviduct <strong>of</strong> recipient mares.Oocytes were obtained from slaughterhousespecimens by opening follicles with a scalpelblade and scraping <strong>the</strong> contents using a bonecurette. Only oocytes having expanded cumuliwere used. Oocytes were cultured in one <strong>of</strong> 3treatments: 1) M199 with 10% fetal calf serum, 5µU FSH/ml and 25 µg/ml gentamycin (mM199)for 24 h; 2) 100% follicular fluid, obtained from apreovulatory follicle aspirated 24 h after hCGadministration, with 25 µg/ml gentamycin (FF) for24 h; or 3) mM199 with 10 µg/ml cycloheximide(CH) for 24 h followed by washing and maturationin mM199 for 24 h. Cycloheximide was used tosuppress meiosis in order to manipulate <strong>the</strong> time <strong>of</strong>onset <strong>of</strong> maturation (Alm and Hinrichs 1996).Oocytes were cultured in microdroplets <strong>of</strong> 10 mlmedium/oocyte under oil in a humidifiedatmosphere <strong>of</strong> 5% CO 2 in air. After culture,oocytes were transferred into both oviducts <strong>of</strong> each<strong>of</strong> 4 mares (a separate treatment per side) viastanding flank laparatomy. Mares wereinseminated 6 h prior to transfer. Oocytes/embryoswere recovered 40–44 h later, following euthanasia<strong>of</strong> <strong>the</strong> mare and removal <strong>of</strong> <strong>the</strong> ovary and oviducts.A total <strong>of</strong> 130 IVM oocytes were transferred.Oocytes were transferred with an intact cumulus,thus <strong>the</strong> state <strong>of</strong> oocyte maturation or degenerationcould not be determined before transfer. Of 100oocytes/embryos recovered, 13 appeared to be <strong>the</strong>recipient mare’s oocytes from previous cycles (iewere flattened, oval and had a pale cytoplasmwithout an intact cytoplasmic membrane)<strong>the</strong>refore, 87 oocytes/embryos recovered wereconsidered to have been from transferred oocytes,giving a 67% recovery <strong>of</strong> transferred oocytes. Onereplicate (9 oocytes) was found to be contaminatedafter recovery, as evidenced by presence <strong>of</strong>bacteria in <strong>the</strong> cytoplasm and white blood cellsattached to <strong>the</strong> zona pellucida, and this replicatewas not included in <strong>the</strong> analysis. Thus, 78 oocyteswere used for evaluation <strong>of</strong> fertilisation rates.All recovered oocytes/embryos were fixed inbuffered formal saline and stained with Hoechst33258 to determine <strong>the</strong>ir chromosomal andnuclear status. A fertilised oocyte was consideredto be an oocyte in any stage from decondensingsperm head to multiple-cell embryo. Thefertilisation rate was determined in 2 ways: as apercentage <strong>of</strong> <strong>the</strong> potentially fertilisable oocytes(ie fertilised oocytes plus oocytes in MII,41