Assisted reproduction technologies have made great progress during the last 15 years in most mammalian species, including humans. Growing evidence indicates that bovine pre-implantation development is a superior model for investigating early human development than the mouse. The purpose of this study was to investigate the effects of two basic culture systems [tissue culture medium (TCM) with 5% CO(2) in air or synthetic oviduct fluid (SOF) with 7% O(2), 88% N(2,) 5% CO(2)] and various protein supplements (serum, bovine serum albumin or polyvinyl alcohol) on the relative abundance of a set of developmentally important gene transcripts in bovine morulae and blastocysts and to compare the results with those for their in-vivo-derived counterparts. The basic culture system including the basic medium composition and oxygen tension had profound effects on the amounts of specific transcripts in bovine embryos, whereas the 'protein source' had only weak effects. Significant differences (P < or = 0.05) in the relative abundance of specific gene transcripts were detected between in-vivo and in-vitro-derived embryos, especially at the morula stage. More differences were found between embryos produced in the TCM system and in-vivo-derived embryos than between SOF-generated embryos and their in-vivo counterparts. No differences were found in the relative abundance of gene transcripts in embryos generated under chemically defined conditions in the two different laboratories. It is concluded that the SOF system provides an environment in which pre-implantation development of bovine embryos is more similar to that occurring in vivo than in the TCM system.

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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 micrograms/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 micrograms/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.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective was to establish an in vitro system in which bovine oocytes can be matured, fertilized, and cultured up to the blastocyst stage without support of serum, BSA, or somatic cells. Media consisted of modified tissue culture medium 199 (mTCM 199) with ovine LH (oLH) for maturation (IVM), experimental alterations of modified defined medium (mDM) for sperm selection and insemination (IVF), and citrate+synthetic oviductal fluid+nonessential amino acids (c-SOF+NEA) for zygote/embryo culture (IVC). Effects of heparin, BSA, polyvinyl alcohol (PVA), penicillamine (P), Hepes, and sodium bicarbonate (NaHCO3) were studied. Results included proportions of oocytes that cleaved by 48 h and that reached morulae by 120 h, blastocysts by 168 h, and expanded blastocysts by 216 h postinsemination (pi). Best results were obtained when the IVF medium included 0.5 mg P+1.0 mg PVA per milliliter with no more than 10 mM Hepes, and when 3.0 mg PVA/ml and 10 mM Hepes were present for IVC. Different concentrations of NaHCO3, up to 50 mM from 25 mM, during IVF did not alter results. Embryos produced in defined conditions yielding the best results remained viable after vitrification as evidenced by continued development in vitro for 96 h postthawing. Bovine oocytes matured in defined medium supplemented with LH were fertilized and cultured up to the blastocyst stage in chemically defined conditions that afforded results comparable to those reported earlier after inclusion of serum, BSA, and/or somatic cells.