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

More documents

Recommendations

Info

Havemeyer Foundation Monograph Series No. 3equine oocytes matured in vitro (Table 1).However, <strong>the</strong> number <strong>of</strong> oocytes that reachedmetaphase II (MII) in vitro was not different whenFCS, PVA and fetuin was added to <strong>the</strong> medium(Table 1). This is in agreement with our previousresults showing similar maturation rates for equineoocytes matured in presence or absence <strong>of</strong> serumand hormones (Landim-Alvarenga and Choi1999).In this experiment <strong>the</strong> time necessary fordigestion <strong>of</strong> 50% <strong>of</strong> <strong>the</strong> zona pellucida (d 50 ) justafter collection was 12.04 min. There was nochange in ZP hardening during <strong>the</strong> culture periodwhen PVA was used. When FCS and Fetuin wereadded to <strong>the</strong> maturation media <strong>the</strong> time necessaryfor digestion <strong>of</strong> 50% <strong>of</strong> <strong>the</strong> zona (d 50 ) decreased inlonger periods <strong>of</strong> culture, however no statisticdifference were noted (Table 2). After 36 h <strong>of</strong>maturation <strong>the</strong> d 50 <strong>of</strong> equine oocytes matured inpresence <strong>of</strong> FCS and fetuin was lower comparedwith <strong>the</strong> group with PVA (Table 2). This is inagreement with <strong>the</strong> results <strong>of</strong> Schroeder et al.(1990) who showed that fetuin inhibited zonapellucida hardening <strong>of</strong> mouse oocytes in a dosedependent way. Also Dell’ Aquila et al. (1998)observed that bovine fetuin inhibited zonapellucida hardening <strong>of</strong> equine oocytes during IVMwith a dose effect.Oocyte maturation in vitro or in vivo isaccompanied by a slow release <strong>of</strong> approximately30–40% <strong>of</strong> <strong>the</strong> cortical granules (Dulcibella et al.1990). However, in vivo, this precocious release<strong>of</strong> cortical granules does not result in zonapellucida modifications. In fact, <strong>the</strong> d 50 measuredin 12 in vivo matured oocytes aspirated frompreovulatory follicles 30 h after hCG injectionwas 9.22 min.The ultrastructural analysis <strong>of</strong> in vivo maturedoocytes showed <strong>the</strong> highest number <strong>of</strong> CC beneath<strong>the</strong> oolema. Similar pattern was observed whenoocytes were matured in vitro for 36 h in presence<strong>of</strong> Fetuin and FCS. However, when maturationoccurs in absence <strong>of</strong> serum, <strong>the</strong> number <strong>of</strong> CC wassmaller. These indicate that in this group aprecocious release <strong>of</strong> CG may happened,justifying <strong>the</strong> high d 50 value observed.We conclude that equine ZP became resistantto lisys by protease following 36 h incubation inserum free media. The addition <strong>of</strong> fetuin or FCS to<strong>the</strong> maturation media reduced <strong>the</strong> d 50 to valuessimilar to <strong>the</strong> ones observed in vivo maturedoocytes. It is likely that <strong>the</strong> problems related toIVF <strong>of</strong> equine oocytes matured in presence <strong>of</strong>serum are not related with ZPH.ACKNOWLEDGEMENTSSupported by PEG - CSU/USA, FAPESP/Braziland FUNDUNESP/ Brazil.REFERENCESChoi, Y.H., Hochi, S., Braun, J. and Oguri, N. (1993)Theriogenol. 40, 959-66.Dell’Aquila, M.E., De Felici, M., Maritato, F.,Lacalandra, G.M. and Minoia, P. (1998) In:<strong>Proceedings</strong> <strong>of</strong> <strong>the</strong> 14th Scientific Meeting <strong>of</strong> <strong>the</strong>European Society <strong>of</strong> Embryo Transfer, Venice, Italy.Abstract. 140.Dell’Aquila, M.E., Felici, M., Massari, S., Maritato, F.and Minoia, P. (1999) Biol. Reprod. 61, 533-540.Doulcibella, T., Kurasawa, S., Rangarjn, S., Kopf, G.S.and Schultz, R.M. (1990) Dev. Biol. 137, 46-55.Landim-Alvarenga, F.C. and Choi, Y.H. (1999)Theriogenol. 51, 383.Schroeder, A.C., Schultz, R.M., Kopf, G.S., Taylor, F.R.,Becker, R.B. and Eppig, J.J. (1990) Biol. Reprod. 43,891-897.Zhang, X., Rutlege, J. and Armstrong, D.T. (1991) Mol.Reprod. Dev. 28, 292-6.27
Equine Embryo TransferPOLYVINYLALCOHOL IS SUPERIOR TO BOVINESERUM ALBUMIN IN EQUINE IVF MEDIUMY. H. Choi, G. E. Seidel, Jr., S. Boyazoglu and E. L. Squires*Centro de Reproducción Equina Doña Pilar, Lincoln, Argentina; *Department <strong>of</strong> Veterinary Physiology andPharmacology, College <strong>of</strong> Veterinary Medicine, Texas A&M University, College Station, Texas, USAINTRODUCTIONSeveral studies on predicting stallion spermfertility have been done using hamster or equineoocytes. However, expenses <strong>of</strong> hamster and equineoocytes for investigating stallion sperm fertility arehigh compared with bovine oocytes, which <strong>of</strong>tencan easily be obtained from local slaughterhouses.Bovine serum albumin is widely used tosupplement capacitation and fertilisation media.However, BSA is contaminated by o<strong>the</strong>r serumcomponents including enzymes, hormones, lipids,and organic and inorganic ions (Bavister 1995). Tomake a defined culture system for examination <strong>of</strong>mechanisms <strong>of</strong> capacitation or fertilisation,polyvinylalcohol (PVA) was used as a substitutefor BSA in one 2 x 2 factorial study usingpenetration <strong>of</strong> zona-free bovine oocytes, and in asecond study using equine oocytes collected fromslaughterhouse-derived ovaries.MATERIALS AND METHODSFor Experiment 1, Bovine oocytes were collectedfrom slaughterhouse-derived ovaries and maturedin TCM-199 with hormones (FSH, LH and E 2 ) and10% oestrous cow serum. After 22 h <strong>of</strong> culture,oocytes were denuded <strong>of</strong> cumulus cells by gentlepipetting in m-PBS plus hyaluronidase and placedin Tyrode’s medium (pH 2.1) for 1–2 min toremove zonae pellucidae. Washed, fresh ejaculatedsperm from 3 stallions were capacitated at 5 x 10 7sperm/ml in TYH medium (modified Krebs-Ringer bicarbonate) plus 0.5 mM 8-bromo-cAMPfor 3.5 h with or without an additional 15 min in100 nM ionomycin, and supplemented withpolyvinylalcohol (PVA; 1 mg/ml) or BSA (4mg/ml). Intraspecies gametes were co-incubated inTYH/PVA or TYH/BSA for 18–20 h at 10 6sperm/ml. For Experiment 2, equine ovaries weresliced near a slaughterhouse, and oocytes werecollected and transported to <strong>the</strong> laboratory insealed 15 ml conical tubes filled with TCM-199plus 10% FCS covered with paraffin oil. Oocyteswere matured as described for bovine oocytes.After 28–30 h maturation, cumulus cells wereremoved and oocytes with a 1st polar body wereused for fertilisation after approximately one-third<strong>of</strong> <strong>the</strong> zona pellucida was removed with a fragment<strong>of</strong> razor blade in protein-free PBS supplementedwith 0.3 M sucrose (Choi et al. 1994). Freshsemen from 2 stallions was mixed and washedtwice (330 x g for 5 min) in TYH/PVA orTYH/BSA. Capacitation <strong>of</strong> stallion sperm waswith 8-bromo cAMP and ionomycin as describedfor Experiment 1, as was IVF. Experiment 2 wasreplicated 3 times. For both experiments, oocyteswere fixed and stained 18–20 h after <strong>the</strong> start <strong>of</strong>IVF and examined for enlarged sperm heads ormale pronucleus formation.RESULTSTreatment <strong>of</strong> stallion sperm with cAMP andionomycin in TYH/PVA resulted in a higherpenetration rate (P
Page 1 and 2: HavemeyerFoundationHavemeyer Founda
Page 3 and 4: © 2001 by R & W Publications (Newm
Page 5 and 6: Equine Embryo TransferChromatin con
Page 7: Equine Embryo TransferEDITORS’ FO
Page 11 and 12: xEquine Embryo Transfer
Page 13 and 14: 2Equine Embryo Transfer
Page 15 and 16: Equine Embryo TransferHYSTEROSCOPIC
Page 17 and 18: Equine Embryo TransferHYSTEROSCOPIC
Page 19 and 20: Equine Embryo TransferUTERINE BLOOD
Page 21 and 22: Equine Embryo Transfer0.900.850.800
Page 27 and 28: Equine Embryo Transferablated 10 da
Page 29 and 30: Equine Embryo TransferREFERENCESBer
Page 31 and 32: Equine Embryo TransferTABLE 1: Chan
Page 33 and 34: Equine Embryo TransferEFFECT OF TEM
Page 35 and 36: Equine Embryo TransferTABLE 2: Effe
Page 37: Equine Embryo TransferEFFECT OF FET
Page 41 and 42: Equine Embryo TransferTHE DEVELOPME
Page 43 and 44: Equine Embryo TransferEMBRYONIC DEV
Page 45 and 46: Equine Embryo Transferbefore matura
Page 47 and 48: Equine Embryo Transfercontaining 10
Page 49 and 50: Equine Embryo TransferOocytes in me
Page 51 and 52: Equine Embryo TransferTABLE 1: Ovul
Page 53 and 54: Equine Embryo Transferdisregarding
Page 55 and 56: Equine Embryo Transferelectric puls
Page 57 and 58: Equine Embryo TransferTABLE 2: Fusi
Page 59 and 60: Equine Embryo Transfercollection; a
Page 63 and 64: Equine Embryo TransferCOMPARISON OF
Page 65 and 66: Equine Embryo TransferCRYOPRESERVAT
Page 67 and 68: Equine Embryo Transferslow-cooling
Page 69 and 70: Equine Embryo TransferTABLE 1: Mean
Page 71 and 72: Equine Embryo TransferPRODUCTION OF
Page 73 and 74: Equine Embryo TransferDOES THE EMBR
Page 75 and 76: Equine Embryo TransferNecrotic rate
Page 77 and 78: Equine Embryo TransferWHEN DO EQUIN
Page 79 and 80: Equine Embryo Transferseems to incr
Page 83 and 84: Equine Embryo Transfer6055ControlIn
Page 85 and 86: Equine Embryo TransferTHE INFLUENCE
Page 87 and 88: Equine Embryo TransferUSE OF BUSERE
Page 89 and 90:
Equine Embryo TransferTABLE 1: Succ
Page 91 and 92:
Equine Embryo TransferTABLE 1: Supe
Page 93 and 94:
Equine Embryo TransferTABLE 1: Freq
Page 95 and 96:
Equine Embryo TransferEMBRYO RECOVE
Page 97 and 98:
Equine Embryo TransferCOMPARISON OF
Page 99 and 100:
Equine Embryo TransferPisa for help
Page 101 and 102:
Equine Embryo TransferThe embryo re
Page 103 and 104:
Equine Embryo Transfervesicles that
Page 105 and 106:
Equine Embryo Transferperipheral se
Page 107 and 108:
Equine Embryo Transferdetected on u
Page 109 and 110:
Equine Embryo TransferHENRIK LEHN-J
Page 111:
Equine Embryo TransferLANDIM-ALVARE
show all

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

Create successful ePaper yourself

Delete template?

Save as template?