IN-VITRO DEVELOPMENTAL COMPETENCE OF IN VITRO-MATURED BOVINE OOCYTES FERTILIZED AND CULTURED IN COMPLETELY DEFINED MEDIA

The objective was to establish an in vitro system in which bovine oocy tes can be matured, fertilized, and cultured up to the blastocyst stag e 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 + synthe tic 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 studi ed. Results included proportions of oocytes that cleaved by 48 h and t hat reached morulae by 120 h, blastocysts by 168 h, and expanded blast ocysts 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 pre sent for IVC. Different concentrations of NaHCO3, up to 50 mM from 25 mM, during IVF did not alter results. Embryos produced in defined cond itions yielding the best results remained viable after vitrification a s evidenced by continued development in vitro for 96 h postthawing. Bo vine oocytes matured in defined medium supplemented with LH were ferti lized and cultured up to the blastocyst stage in chemically defined co nditions that afforded results comparable to those reported earlier af ter inclusion of serum, BSA, and/or somatic cells.