Havemeyer Foundati<strong>on</strong> M<strong>on</strong>ograph Series No. 3Didi<strong>on</strong>, B.A., Pomp, D., Martin, M.J., Homanics, G.E.and Markert, C.L. (1990) Observati<strong>on</strong>s <strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g>cooling and cryopreservati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> pig oocytes at <str<strong>on</strong>g>the</str<strong>on</strong>g>germinal vesicle stage. J. anim. Sci. 68, 2803-2810.Guignot, F., Bezard, J. and Palmer, E. (1999) Effect <str<strong>on</strong>g>of</str<strong>on</strong>g>time during transport <str<strong>on</strong>g>of</str<strong>on</strong>g> excised mare ovaries <strong>on</strong>oocyte recovery rate and quality after in vitromaturati<strong>on</strong>. Theriogenol. 52, 757-766.Hinrichs, K., Schmidt, A.L., Friedman, P.P., Selgrath, J.P.and Martin, M.G. (1993a) In vitro maturati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g>horse oocytes: characterizati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> chromatinc<strong>on</strong>figurati<strong>on</strong> using fluorescence microscopy. Biol.Reprod. 48, 363-370.Hinrichs, K., Schmidt, A.L. and Selgrath, J.P. (1993b)Atlas <str<strong>on</strong>g>of</str<strong>on</strong>g> chromatin c<strong>on</strong>figurati<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> germinalvesicle-stage and maturing horse oocytes. Equinevet. J. Suppl. 15, 60-63.Moodie, G.R. and Graham, E.F. (1989) The effect <str<strong>on</strong>g>of</str<strong>on</strong>g>incubating sheep ovaries for various times andtemperatures <strong>on</strong> oocyte maturati<strong>on</strong> rates in vitro.Biol. Reprod. 40, abstract 53.Moor, R.M. and Crosby, I.M. (1985) Temperatureinducedabnormalities in sheep oocytes duringmaturati<strong>on</strong>. J. reprod. Fertil. 75, 467-473.Richard, F.J. and Sirard, M.A. (1996) Effects <str<strong>on</strong>g>of</str<strong>on</strong>g> harvestmethods <str<strong>on</strong>g>of</str<strong>on</strong>g> bovine oocytes cocultured with follicularhemisecti<strong>on</strong>s in vitro <strong>on</strong> nuclear maturati<strong>on</strong>.Theriogenol. 46, 1243-1250.Schroeder, A.C., Johnst<strong>on</strong>, D. and Eppig, J.J. (1991)Reversal <str<strong>on</strong>g>of</str<strong>on</strong>g> postmortem degenerati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> mouseoocytes during meiotic maturati<strong>on</strong> in vitro. J. exp.Zool. 258, 240-245.Shabpareh, V., Squires, E.L., Seidel, G.E. and Jasko, D.J.(1993) Methods for collecting and maturing equineoocytes in vitro. Theriogenol. 40, 1161-1175.Zer<strong>on</strong>, Y., Pearl, M., Borochov, A. and Arav, A. (1999)Kinetic and temporal factors influence chillinginjury to germinal vesicle and mature bovineoocytes. Cryobiol. 38, 35-42.25
Equine Embryo TransferEFFECT OF FETUIN ON THE ZONA HARDENINGAND CORTICAL GRANULES DISTRIBUTION IN<strong>EQUINE</strong> OOCYTES MATURED IN VITROF. C. Landim-Alvarenga, S. E. A. Boyazoglu*, G. E. Jr. Seidel † and E. L. Squires †Department <str<strong>on</strong>g>of</str<strong>on</strong>g> Animal Reproducti<strong>on</strong> and Veterinary Radiology, F.M.V.Z. - UNESP, Botucatu, SP,18618.000, Brazil; *Sport Horse Research Center, Faculty <str<strong>on</strong>g>of</str<strong>on</strong>g> Veterinary Medicine – Perugia, 06126,Italy; † ARBL, Colorado State University, Fort Collins, Colorado 80523, USAFully grown oocytes from many mammals canundergo sp<strong>on</strong>taneous meiotic maturati<strong>on</strong> when<str<strong>on</strong>g>the</str<strong>on</strong>g>y are liberated from <str<strong>on</strong>g>the</str<strong>on</strong>g>ir follicles and culturedin vitro. However, <str<strong>on</strong>g>the</str<strong>on</strong>g> z<strong>on</strong>a pellucida becomesresistant to chymotrypsin digesti<strong>on</strong> or ‘hardened’,when sp<strong>on</strong>taneous maturati<strong>on</strong> occurs in serumfreemedium (Zhang et al. 1991). Schroeder et al.(1990) described that fetuin, a comp<strong>on</strong>ent <str<strong>on</strong>g>of</str<strong>on</strong>g> FetalBovine Serum (FBS), inhibits z<strong>on</strong>a pellucidahardening during oocyte maturati<strong>on</strong>. Dell’ Aquilaet al. (1999) studied <str<strong>on</strong>g>the</str<strong>on</strong>g> effect <str<strong>on</strong>g>of</str<strong>on</strong>g> additi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> fetuinin maturati<strong>on</strong> media <str<strong>on</strong>g>of</str<strong>on</strong>g> equine oocytes in presence<str<strong>on</strong>g>of</str<strong>on</strong>g> equine follicular fluid, oestrous mare serum,equine fetal serum and BSA. The results showedthat fetuin reduces z<strong>on</strong>a hardening, but it does notinfluence sperm penetrati<strong>on</strong> after IVF. However,<str<strong>on</strong>g>the</str<strong>on</strong>g>re is a lack <str<strong>on</strong>g>of</str<strong>on</strong>g> informati<strong>on</strong> <strong>on</strong> z<strong>on</strong>a-hardening <str<strong>on</strong>g>of</str<strong>on</strong>g>in vivo matured oocytes. Also, <str<strong>on</strong>g>the</str<strong>on</strong>g>re is no papercorrelating z<strong>on</strong>a hardening and cortical granulesdistributi<strong>on</strong> during different culture periods inequine oocytes. So, <str<strong>on</strong>g>the</str<strong>on</strong>g> objective <str<strong>on</strong>g>of</str<strong>on</strong>g> this study wasto compare z<strong>on</strong>a hardening <str<strong>on</strong>g>of</str<strong>on</strong>g> in vitro and in vivomatured oocytes and to determine whe<str<strong>on</strong>g>the</str<strong>on</strong>g>rexposure <str<strong>on</strong>g>of</str<strong>on</strong>g> equine oocytes to serum-free mediumc<strong>on</strong>taining fetuin would affect ZPH and corticalgranule (CC) distributi<strong>on</strong>.MATERIALAND METHODSOocytes were collected from slaughterhouseovaries according to <str<strong>on</strong>g>the</str<strong>on</strong>g> method described by Choiet al. (1993) and matured in TCM 199 with Earle’ssalt (Sigma M7529, M<strong>on</strong>tana, USA) supplementedwith pyruvate, FSH and Oestradiol 17ß in <strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g><str<strong>on</strong>g>the</str<strong>on</strong>g> following treatments:Group 1 (c<strong>on</strong>trol): maturati<strong>on</strong> medium c<strong>on</strong>taining10% FCS;Group 2: maturati<strong>on</strong> medium + 1 mg/mlpolivinylalcohol (PVA) andGroup 3: maturati<strong>on</strong> medium + 1 mg/ml Fetuin.Fifteen to 20 oocytes were cultured in100 µl drops covered with washed, equilibratedparaffin oil. All oocytes were incubated for 36–40h at 38.5°C in 5% CO 2 in air. Five replicates wereused.In vivo (n=12) matured oocytes were obtainedfrom preovulatory follicles. Normal cycling mareswere treated with hCG when <strong>on</strong>e follicle reached35 mm in diameter. After 30 h a transvaginalaspirati<strong>on</strong> was performed to collect <str<strong>on</strong>g>the</str<strong>on</strong>g> oocyte.After maturati<strong>on</strong> <str<strong>on</strong>g>the</str<strong>on</strong>g> oocytes were striped <str<strong>on</strong>g>of</str<strong>on</strong>g>cumulus cells using a fine bore pipette in 1% BSAmodified Dulbecco’s PBS (DPBS) + hyalur<strong>on</strong>idasetype V (300 iu/ml). Totally denuded oocytes werewashed <strong>on</strong>ce in DPBS and transferred to a soluti<strong>on</strong>c<strong>on</strong>taining 0.3% protease (Pr<strong>on</strong>ase – Sigma,M<strong>on</strong>tana, USA) in DPBS. The 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 pellucida was estimate by <str<strong>on</strong>g>the</str<strong>on</strong>g> time necessaryfor digesti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> 50% <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> ZP (d 50 ). The z<strong>on</strong>a freeoocytes were <str<strong>on</strong>g>the</str<strong>on</strong>g>n stained with 45% acetic orceinfor evaluati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> nuclear status. Effects <str<strong>on</strong>g>of</str<strong>on</strong>g>maturati<strong>on</strong> systems were analysed by ANOVA.Cortical granules distributi<strong>on</strong> was analysedusing Transmissi<strong>on</strong> Electr<strong>on</strong> Microscopy insamples <str<strong>on</strong>g>of</str<strong>on</strong>g> 5 oocytes taken just after collecti<strong>on</strong> andafter 15, 24 and 36 h <str<strong>on</strong>g>of</str<strong>on</strong>g> maturati<strong>on</strong> in eachtreatment. A group <str<strong>on</strong>g>of</str<strong>on</strong>g> 4 in vivo matured oocyteswas also used.RESULTS AND DISCUSSIONThe additi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> FCS to <str<strong>on</strong>g>the</str<strong>on</strong>g> maturati<strong>on</strong> mediasignificantly increase cumulus cell expansi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g>26
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