PRODUCTION OF VIABLE BOVINE BLASTOCYSTS IN DEFINED IN-VITRO CONDITIONS

Our purpose was to obtain viable blastocysts via in vitro maturation, fertilization, and culture (IVMFC) in serum- and BSA-free media (defin ed conditions) and to document viability by pregnancy initiation follo wing embryo transfer (ET). Oocytes were matured in modified TCM 199 (m TCM 199) with 100 mu g/ml ovine (o)LH, inseminated in TALP- or defined medium (DM)-based media, and cultured up to 9 days in synthetic ovidu ctal fluid (SOF) prepared with 6 mg/ml polyvinyl alcohol (PVA) instead of BSA and buffered with 25 mM HEPES with experimental modifications. Modifications for embryo culture included supplementation with Minimu m Essential Medium amino acids (MEM), Minimum Essential Medium nonesse ntial amino acids (NEA), the combination of MEM and NEA, citrate (c; 0 .5 mM), glutamine (1 mM), or combinations of these. Proportions of imm ature oocytes selected for IVM that cleaved (IVF) and that reached the blastocyst stage in SOF were 66.3% and 10.9%, respectively. Supplemen tation of SOF with citrate and nonessential amino acids (i.e., c-SOF NEA) enabled 85.1% cleavage and 42.6% blastocyst development of oocyt es selected for IVM. In conjunction with IVM in mTCM 199 plus 100 mu g /ml oLH and IVC in c-SOF + NEA efforts to eliminate protein from the f ertilization medium revealed modified DM (mDM) prepared with PVA inste ad of BSA to be superior to TALP prepared with PVA; IVMFC data for bla stocyst development were 27.4% vs. 18.2% (p < 0.05), respectively. The use of mDM for sperm preparation and IVF yielded comparable blastocys t development when either BSA or PVA was included. One of two recipien ts receiving single Day-6 embryos fertilized in the presence of BSA de livered a 36.5-kg bull calf on gestation Day 282. This report document s the complete development of immature bovine oocytes to blastocysts v ia IVMFC in defined media. Viability of two uterine-stage embryos prod uced in defined conditions was evidenced by initiation of pregnancies in two additional ET recipients. Both of these pregnancies resulted in abortions that could be attributed, at least in part, to dysfunctiona l conditions of the recipients.