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 (defined conditions) and to document viability by pregnancy initiation following embryo transfer (ET). Oocytes were matured in modified TCM 199 (mTCM 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 oviductal 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 Minimum Essential Medium amino acids (MEM), Minimum Essential Medium nonessential amino acids (NEA), the combination of MEM and NEA, citrate (c; 0.5 mM), glutamine (1 mM), or combinations of these. Proportions of immature oocytes selected for IVM that cleaved (IVF) and that reached the blastocyst stage in SOF were 66.3% and 10.9%, respectively. Supplementation of SOF with citrate and nonessential amino acids (i.e., c-SOF + NEA) enabled 85.1% cleavage and 42.6% blastocyst development of oocytes 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 fertilization medium revealed modified DM (mDM) prepared with PVA instead of BSA to be superior to TALP prepared with PVA; IVMFC data for blastocyst development were 27.4% vs. 18.2% (p < 0.05), respectively. The use of mDM for sperm preparation and IVF yielded comparable blastocyst development when either BSA or PVA was included. One of two recipients receiving single Day-6 embryos fertilized in the presence of BSA delivered a 36.5-kg bull calf on gestation Day 282. This report documents the complete development of immature bovine oocytes to blastocysts via IVMFC in defined media. Viability of two uterine-stage embryos produced 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 dysfunctional conditions of the recipients.