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

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Havemeyer Foundation Monograph Series No. 3EFFECT OF INVASIVE ADENYLATE CYCLASEDURING OOCYTE MATURATION ON DEVELOPMENTOF EQUINE EMBRYOS FOLLOWING ICSIL. J. Maclellan, M. M. Sims and E. L SquiresAnimal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado80523, USAInvasive adenylate cyclase (iAC), a toxin producedby <strong>the</strong> bacterium Bordetella pertussis, can beinternalised by mammalian cells resulting inincreased production <strong>of</strong> cAMP from <strong>the</strong> cells ownpool <strong>of</strong> adenosine 5-triphosphate (ATP) (Confer etal. 1984). During oocyte collection procedures<strong>the</strong>re is a significant decrease in intracellular levels<strong>of</strong> cAMP [cAMPi] in cumulus oocyte complexes.Including iAC in <strong>the</strong> collection medium canprevent this [cAMPi] decrease. Culturing bovineoocytes with low concentrations <strong>of</strong> invasiveadenylate cyclase during IVM can stimulatenuclear maturation while high concentrationsinhibit nuclear maturation. In addition, lowconcentrations <strong>of</strong> iAC in maturation media resultin increased rates <strong>of</strong> cleavage and increased rates<strong>of</strong> development to <strong>the</strong> blastocysts post IVF(Luciano et al. 1999).The objective <strong>of</strong> <strong>the</strong> experiment was todetermine <strong>the</strong> effect <strong>of</strong> invasive adenylate cyclaseduring in vitro maturation <strong>of</strong> equine oocytes onnuclear maturation and embryo developmentfollowing ICSI.Equine oocytes were collected within 3 h <strong>of</strong>slaughtering mares in three replicates. Oocyteswere obtained by slicing ovaries and washing inTCM-199 plus 0.1% BSA with or without 0.01mM iAC and 0.5 mM 3-isobutyl 1-methylxanthine. Cumulus oocyte complexes(COC) were characterised as: 1) compact cumulus(CP) – having 3 or more layers <strong>of</strong> cumulus cellstightly adhered toge<strong>the</strong>r, completely surrounding<strong>the</strong> oocyte; or 2) expanded cumulus (EX) – havinga completely expanded cumulus with a cellulargelatinous cloud around <strong>the</strong> oocyte. Oocytes weretransported in a Minitub portable incubator to <strong>the</strong>laboratory in 6 ml Falcon tubes at 38.5ºC in mediaequilibrated under 6% CO 2 in air. Oocytes were<strong>the</strong>n transferred to 4-well plates for <strong>the</strong> remainder<strong>of</strong> <strong>the</strong> maturation period. There were 4 treatmentgroups: control compact (n=104) and controlexpanded COCs (n=56) cultured in TCM-199 with0.6% BSA and hormones (1 µg/ml b-LH, 15 ng/mlo-FSH, 1 µg/ml E2 and 500 ng/ml progesterone);iAC compact (n=95), and iAC expanded COCs(n=52), cultured in TCM-199 with 0.6% BSA and0.01 mM iAC.A sample <strong>of</strong> oocytes was removed from eachgroup at 24 h for nuclear staining with acetoorcein.After maturation for 34–36 h oocytes weredenuded by manual pipetting in <strong>the</strong> presence <strong>of</strong>300 iu hyalauronidase/ml. Frozen thawed spermwas prepared over a discontinuous 90/45% Percollgradient and re-suspended in hepes-buffered SOFTABLE 1: Effects <strong>of</strong> iAC during oocyte maturation on subsequent developmentCleaved 2-8 cell Morulae Blastocyst Fragmentn (%) n (%) n (%) n (%) n (%)Control compact 24/68 (35) 20(30) 4(6) 0 7(10)Control expanded 7/32 (22) 7(22) 0 0 4(13)0.01mM iAC compact 18/57(32) 12(21) 4(7) 2(4) 4(7)0.01mM iAC expanded 8/31(26) 7(23) 1(3) 0 6(19)35
Equine Embryo Transfercontaining 10% polyvinylpyrrolidone (PVP, MW360,000). Oocytes were injected with a singlespermatozoon. All oocytes were activated with 10µM calcium ionophore A23187 for 5 min andde-activated in SOF containing 20 mg/ml BSAbefore being transferred to culture dishescontaining modified syn<strong>the</strong>tic oviduct fluid (Laneand Gardner 1997).Averaged over compact and expandedcumulus groups, oocytes in <strong>the</strong> iAC group tendedto be more advanced in nuclear maturation at 24 h;32/49 (65%) in MI or MII for iAC vs 12/30 (40%)for control oocytes (P
Page 1 and 2: HavemeyerFoundationHavemeyer Founda
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Page 33 and 34: Equine Embryo TransferEFFECT OF TEM
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Page 37 and 38: Equine Embryo TransferEFFECT OF FET
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Page 41 and 42: Equine Embryo TransferTHE DEVELOPME
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Page 75 and 76: Equine Embryo TransferNecrotic rate
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Page 85 and 86: Equine Embryo TransferTHE INFLUENCE
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Equine Embryo TransferPisa for help
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Equine Embryo TransferHENRIK LEHN-J
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Equine Embryo TransferLANDIM-ALVARE
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