Production of viable bovine blastocysts in defined in vitro conditions

Abstract

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)

Effects of Glutamine, Glycine and Taurine on the Development of In Vitro Fertilized Bovine Zygotes in a Chemically Defined Medium.

“…In the present study, however, no beneficial effect of glutamine in combination with essential amino acids for basal medium Eagle (EAA-BME) and nonessential amino acids for minimum essential medium (NEAA-MEM) was observed even in the absence of glucose. This result supports the recent finding that the addition of 1 mM glutamine singly or in combination with EAA-MEM and/or NEAA-MEM, does not improve bovine embryo development [10,12,33]. Further experiments are needed to determine the optimal concentrations of glutamine for in vitro culture and its role in the development of bovine embryos, since it has been demonstrated that bovine embryos utilize glutamine as an energy source throughout their preimplantation development [25], and that the response of mouse embryos to glutamine depended on the concentration of NaCl in the culture medium [11].…”

“…The effects of glutamine and glycine on bovine embryonic development have been investigated by several groups, however, the supplementation of media with glutamine [10,12,20,23,33] or glycine [12,15,21,28] has given inconsistent results. One of the reasons is that the culture conditions used in these studies were variable: complex vs. simple media, with vs. without other amino acids, presence vs. absence of bovine serum albumin (BSA), and so on.…”

Differences in developmental competence and gene expression profiles between buffalo (Bubalus bubalis) preimplantation embryos cultured in three different embryo culture media

“…Chemically defined conditions without serum or serum derived proteins allow more precise observation of effects of growth or other embryo growth factors in a given medium (Giles and Foote 1997;Sirisathien et al 2003;Oyamada et al 2004). It was demonstrated that chemically defined medium supported bovine zygote development to blastocysts with varying rates of success (Keskintepe et al 1995;Keskintepe and Brackett 1996;Lim et al 2007).…”

Effects of glucose and protein sources on bovine embryo development in vitro

“…Keskintepe et al, 1995;Takahashi et al, 1996;Matsui et al, 1997 , protein supplemen-Ž tation has been shown to be beneficial for embryo development in vitro Takahashi and . First, 1992;Carolan et al, 1995 .…”