Havemeyer Foundati<strong>on</strong> M<strong>on</strong>ograph 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 <str<strong>on</strong>g>of</str<strong>on</strong>g> Physiology and Pharmacology; *Department <str<strong>on</strong>g>of</str<strong>on</strong>g> Large Animal Medicine and Surgery,College <str<strong>on</strong>g>of</str<strong>on</strong>g> Veterinary Medicine, Texas A & M University, College Stati<strong>on</strong>, Texas 77843-4475, USAIn vitro-matured (IVM) oocytes are comm<strong>on</strong>lyused for experimentati<strong>on</strong> involving in vitr<str<strong>on</strong>g>of</str<strong>on</strong>g>ertilisati<strong>on</strong> (IVF). However, <str<strong>on</strong>g>the</str<strong>on</strong>g> fertilisati<strong>on</strong>potential <str<strong>on</strong>g>of</str<strong>on</strong>g> IVM oocytes is unknown. The majorbarrier to IVF in <str<strong>on</strong>g>the</str<strong>on</strong>g> horse appears to bepenetrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> sperm through <str<strong>on</strong>g>the</str<strong>on</strong>g> z<strong>on</strong>a pellucida. If<str<strong>on</strong>g>the</str<strong>on</strong>g> z<strong>on</strong>a is opened by microdissecti<strong>on</strong> or z<strong>on</strong>adrilling, IVF rates are high (Choi et al. 1994; Li etal. 1995). It is possible that in vitro maturati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g>oocytes is associated with hardening <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> z<strong>on</strong>apellucida which precludes penetrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> sperm.Hardening <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> z<strong>on</strong>a due to premature release <str<strong>on</strong>g>of</str<strong>on</strong>g>cortical granules has been documented duringIVM in o<str<strong>on</strong>g>the</str<strong>on</strong>g>r species under specific culturec<strong>on</strong>diti<strong>on</strong>s. The ability <str<strong>on</strong>g>of</str<strong>on</strong>g> IVM oocytes to allowsperm penetrati<strong>on</strong> must be ascertained before <str<strong>on</strong>g>the</str<strong>on</strong>g>ymay be used effectively for research in IVF. Only<strong>on</strong>e previous study (Zhang et al. 1989) hasdocumented fertilisati<strong>on</strong> and development <str<strong>on</strong>g>of</str<strong>on</strong>g> IVMoocytes after transfer to mares. In that report, 4transfers were performed. Of 29 oocytestransferred, 5 c<strong>on</strong>firmed blastocysts wererecovered. The objective <str<strong>on</strong>g>of</str<strong>on</strong>g> our study was todetermine <str<strong>on</strong>g>the</str<strong>on</strong>g> in vivo fertilisati<strong>on</strong> rate <str<strong>on</strong>g>of</str<strong>on</strong>g> oocytesmatured in vitro in 3 different media andtransferred to <str<strong>on</strong>g>the</str<strong>on</strong>g> oviduct <str<strong>on</strong>g>of</str<strong>on</strong>g> recipient mares.Oocytes were obtained from slaughterhousespecimens by opening follicles with a scalpelblade and scraping <str<strong>on</strong>g>the</str<strong>on</strong>g> c<strong>on</strong>tents using a b<strong>on</strong>ecurette. Only oocytes having expanded cumuliwere used. Oocytes were cultured in <strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> 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 hCGadministrati<strong>on</strong>, with 25 µg/ml gentamycin (FF) for24 h; or 3) mM199 with 10 µg/ml cycloheximide(CH) for 24 h followed by washing and maturati<strong>on</strong>in mM199 for 24 h. Cycloheximide was used tosuppress meiosis in order to manipulate <str<strong>on</strong>g>the</str<strong>on</strong>g> time <str<strong>on</strong>g>of</str<strong>on</strong>g><strong>on</strong>set <str<strong>on</strong>g>of</str<strong>on</strong>g> maturati<strong>on</strong> (Alm and Hinrichs 1996).Oocytes were cultured in microdroplets <str<strong>on</strong>g>of</str<strong>on</strong>g> 10 mlmedium/oocyte under oil in a humidifiedatmosphere <str<strong>on</strong>g>of</str<strong>on</strong>g> 5% CO 2 in air. After culture,oocytes were transferred into both oviducts <str<strong>on</strong>g>of</str<strong>on</strong>g> each<str<strong>on</strong>g>of</str<strong>on</strong>g> 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<str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> mare and removal <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> ovary and oviducts.A total <str<strong>on</strong>g>of</str<strong>on</strong>g> 130 IVM oocytes were transferred.Oocytes were transferred with an intact cumulus,thus <str<strong>on</strong>g>the</str<strong>on</strong>g> state <str<strong>on</strong>g>of</str<strong>on</strong>g> oocyte maturati<strong>on</strong> or degenerati<strong>on</strong>could not be determined before transfer. Of 100oocytes/embryos recovered, 13 appeared to be <str<strong>on</strong>g>the</str<strong>on</strong>g>recipient mare’s oocytes from previous cycles (iewere flattened, oval and had a pale cytoplasmwithout an intact cytoplasmic membrane)<str<strong>on</strong>g>the</str<strong>on</strong>g>refore, 87 oocytes/embryos recovered werec<strong>on</strong>sidered to have been from transferred oocytes,giving a 67% recovery <str<strong>on</strong>g>of</str<strong>on</strong>g> transferred oocytes. Onereplicate (9 oocytes) was found to be c<strong>on</strong>taminatedafter recovery, as evidenced by presence <str<strong>on</strong>g>of</str<strong>on</strong>g>bacteria in <str<strong>on</strong>g>the</str<strong>on</strong>g> cytoplasm and white blood cellsattached to <str<strong>on</strong>g>the</str<strong>on</strong>g> z<strong>on</strong>a pellucida, and this replicatewas not included in <str<strong>on</strong>g>the</str<strong>on</strong>g> analysis. Thus, 78 oocyteswere used for evaluati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> fertilisati<strong>on</strong> rates.All recovered oocytes/embryos were fixed inbuffered formal saline and stained with Hoechst33258 to determine <str<strong>on</strong>g>the</str<strong>on</strong>g>ir chromosomal andnuclear status. A fertilised oocyte was c<strong>on</strong>sideredto be an oocyte in any stage from dec<strong>on</strong>densingsperm head to multiple-cell embryo. Thefertilisati<strong>on</strong> rate was determined in 2 ways: as apercentage <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> potentially fertilisable oocytes(ie fertilised oocytes plus oocytes in MII,41
Equine Embryo Transferdisregarding degenerating oocytes) and as apercentage <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> total oocytes recovered.The fertilisati<strong>on</strong> rates for potentiallyfertilisable oocytes for <str<strong>on</strong>g>the</str<strong>on</strong>g> mM199, FF, and CHgroups (3, 3 and <strong>on</strong>e replicates, respectively) were11/15 (73%); 22/30 (73%) and 6/12 (50%),respectively. The fertilisati<strong>on</strong> rates based <strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g>total oocytes recovered for each treatment were11/25 (44%), 22/37 (59%) and 6/16 (38%),respectively. The overall fertilisati<strong>on</strong> rate forpotentially fertilisable oocytes was 68% (39/57)and <str<strong>on</strong>g>the</str<strong>on</strong>g> fertilisati<strong>on</strong> rate for all oocytes recoveredwas 50% (39/78).One recovered oocyte was in Anaphase II witha dec<strong>on</strong>densing sperm head, and <strong>on</strong>e was inmitosis <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> first divisi<strong>on</strong>; <str<strong>on</strong>g>the</str<strong>on</strong>g>se were groupedwith <str<strong>on</strong>g>the</str<strong>on</strong>g> cells having 2 pr<strong>on</strong>uclei. Thus, <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>fertilised oocytes recovered, 20/39 (51%) had 2pr<strong>on</strong>uclei, 13/39 (33%) were 2-cell embryos, and6/39 (15%) were 3- to 4- cell embryos. There wasa significant difference in distributi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> embryostage am<strong>on</strong>g maturati<strong>on</strong> treatments. Theproporti<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> fertilised oocytes that had cleavedto 2 or more - cell embryos in <str<strong>on</strong>g>the</str<strong>on</strong>g> mM199 and CHgroup were both significantly higher than that for<str<strong>on</strong>g>the</str<strong>on</strong>g> FF group (9/11, 82%; 5/6, 83%; and 6/22,27%, respectively; P