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

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