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

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

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Equine Embryo TransferEFFECT OF TEMPERATURE AND HOLDING TIME ON<strong>EQUINE</strong> OOCYTE CHROMATIN CONFIGURATIONH. G. Pedersen, E. E. Telfer* and E. D. Wats<strong>on</strong>Department <str<strong>on</strong>g>of</str<strong>on</strong>g> Veterinary Clinical Studies, University <str<strong>on</strong>g>of</str<strong>on</strong>g> Edinburgh, Easter Bush Veterinary Centre, Roslin,Midlothian EH25 9RG; *Institute <str<strong>on</strong>g>of</str<strong>on</strong>g> Cell and Molecular Biology, University <str<strong>on</strong>g>of</str<strong>on</strong>g> Edinburgh, Darwin Building,West Mains Road, Edinburgh EH9 3JG, UKINTRODUCTIONEquine oocytes are <str<strong>on</strong>g>of</str<strong>on</strong>g>ten obtained from abattoirsand a c<strong>on</strong>siderable period <str<strong>on</strong>g>of</str<strong>on</strong>g> time <str<strong>on</strong>g>of</str<strong>on</strong>g>ten elapsesfrom excisi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> ovaries until recovery <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>oocytes. The effect <str<strong>on</strong>g>of</str<strong>on</strong>g> time and temperature couldpotentially affect <str<strong>on</strong>g>the</str<strong>on</strong>g> resulting in vitro maturati<strong>on</strong>rates but differing results have been obtained in <str<strong>on</strong>g>the</str<strong>on</strong>g>different species studied. In a study in horses,storage <str<strong>on</strong>g>of</str<strong>on</strong>g> ovaries for 6–8 h was found not to affectoocyte maturati<strong>on</strong> rates compared to oocytes fromovaries stored for 1.5–4 h (Guignot et al. 1999).Ano<str<strong>on</strong>g>the</str<strong>on</strong>g>r study reported no effect <strong>on</strong> maturati<strong>on</strong>rates after storage time <str<strong>on</strong>g>of</str<strong>on</strong>g> up to 15 h compared to3–9 h (Delcampo et al. 1995). The same c<strong>on</strong>clusi<strong>on</strong>was reached after comparis<strong>on</strong>s were made betweenstoring <str<strong>on</strong>g>the</str<strong>on</strong>g> equine oocytes at 5–6, 6–7 and 7–8 h,and fur<str<strong>on</strong>g>the</str<strong>on</strong>g>rmore, no difference was found incumulus morphology (Shabpareh et al. 1993). Byc<strong>on</strong>trast, storing ovine oocytes for 4, 8 and 24 h at5ºC, 22ºC or 37ºC, resulted in a negative effect <strong>on</strong><str<strong>on</strong>g>the</str<strong>on</strong>g> oocytes’ capacity to reach Metaphase II.Storing ovine oocytes at 22ºC affected <str<strong>on</strong>g>the</str<strong>on</strong>g> oocytesless than at 5ºC and 37ºC (Moodie and Graham1989) but ano<str<strong>on</strong>g>the</str<strong>on</strong>g>r study found that exposing bovinegerminal vesicle oocytes to temperatures below23°C reduced <str<strong>on</strong>g>the</str<strong>on</strong>g>ir viability as determined bymembrane integrity (Zer<strong>on</strong> et al. 1999). Thedevelopmental capacity <str<strong>on</strong>g>of</str<strong>on</strong>g> murine oocytes to <str<strong>on</strong>g>the</str<strong>on</strong>g> 2cell stage embryo following storage in <str<strong>on</strong>g>the</str<strong>on</strong>g> ovariesfor up 6 h was not different from c<strong>on</strong>trol oocytes,but storage for 9–12 h was detrimental todevelopment (Schroeder et al. 1991).The aim <str<strong>on</strong>g>of</str<strong>on</strong>g> this study was to investigate <str<strong>on</strong>g>the</str<strong>on</strong>g>effect <str<strong>on</strong>g>of</str<strong>on</strong>g> holding temperature and time <strong>on</strong> equineoocyte chromatin c<strong>on</strong>figurati<strong>on</strong> and cumulusmorphology immediately after recovery from <str<strong>on</strong>g>the</str<strong>on</strong>g>follicle.MATERIALS AND METHODSOvaries were obtained from horses and p<strong>on</strong>iesafter slaughter or after ovariectomy. Theexteriorised ovaries were kept at 20–30ºC or35–37ºC in M199 with Hanks salts and 25 mMHepes for 0.5–24 h. Processed follicles rangedfrom 2–60 mm in diameter. The inner wall <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>follicle was scraped with a b<strong>on</strong>e curette to release<str<strong>on</strong>g>the</str<strong>on</strong>g> cumulus oophorus with <str<strong>on</strong>g>the</str<strong>on</strong>g> oocyte. Afterexaminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> cumulus oophorus, <str<strong>on</strong>g>the</str<strong>on</strong>g> oocytewas denuded, fixed and stained with 2.5 µg/mlHoechst stain 33258 (bisBenzimide). Cumulusoophorus morphology was classified ei<str<strong>on</strong>g>the</str<strong>on</strong>g>r ascompact (CP), expanded (EX), or denuded using<str<strong>on</strong>g>the</str<strong>on</strong>g> criteria <str<strong>on</strong>g>of</str<strong>on</strong>g> (Hinrichs et al. 1993a). The oocytechromatin c<strong>on</strong>figurati<strong>on</strong>s were classified asfluorescent nucleus (FN), loosely c<strong>on</strong>densedchromatin (LCC), c<strong>on</strong>densed chromatin (CC),metaphase, or abnormal according to criteria <str<strong>on</strong>g>of</str<strong>on</strong>g>(Hinrichs et al. 1993b). The study c<strong>on</strong>sisted <str<strong>on</strong>g>of</str<strong>on</strong>g> 4experiments.Experiment 1: The effect <str<strong>on</strong>g>of</str<strong>on</strong>g> temperature <strong>on</strong>chromatin c<strong>on</strong>figurati<strong>on</strong>Oocytes from ovaries stored at 20–30ºC (n=14) or35–37ºC (n=59) were used. To avoid <str<strong>on</strong>g>the</str<strong>on</strong>g>c<strong>on</strong>founding effect <str<strong>on</strong>g>of</str<strong>on</strong>g> time, <strong>on</strong>ly oocytes that hadbeen sitting in <str<strong>on</strong>g>the</str<strong>on</strong>g> follicles for less than 3 h wereselected.Experiment 2: The effect <str<strong>on</strong>g>of</str<strong>on</strong>g> time <strong>on</strong> chromatinc<strong>on</strong>figurati<strong>on</strong>Oocytes (n=222) were stored in <str<strong>on</strong>g>the</str<strong>on</strong>g> follicles for0–1, 1–2, 2–3, 3–4, 4–6, 6–8 and 8–12 h afterexteriorisati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> ovary.22

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